Method for evaluating the ability of a compound to inhibit the protoporphyrinogen oxidase activity

ABSTRACT

The present invention provides a method for evaluating the ability of a compound to inhibit protoporphyrinogen oxidase activity, which comprises the steps of (1) culturing a transformant expressing a heterologous protoporphyrinogen oxidase (PPO) gene in a medium containing substantially no protoheme compounds in the presence and absence of a test compound to measure the growth rate of the transformant, wherein the transformant is a host cell deficient in PPO activity which is transformed with the heterologous PPO gene, and (2) determining the ability of the compound to inhibit the protoporphyrinogen oxidase activity by comparing growth rates.

This application is a divisional of U.S. application Ser. No. 09/289,180filed on Apr. 9, 1999, now U.S. Pat. No. 6,472,164.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for evaluating the ability ofa compound to inhibit the protoporphyrinogen oxidase activity.

2. Description of the Related Art

Plants, animals and microorganisms possess the porphyrin biosyntheticsystem starting with 5-aminolevulinic acid and produce protoporphyrinwhich is a precursor for the heme biosynthesis and the like. An enzymewhich catalyses a reaction oxidizing protoporphyrinogen at the finalstage of the porphyrin biosynthesis to produce protoporphyrin isprotoporphyrinogen oxidase (protoporphyrinogen IX oxidase; EC1.3.3.4)(hereinafter referred to as “PPO”)(R. J. Porra and J. E. Falk,(1964) Biochem. J., Vol.90, pp.69-75).

The PPO activity has the influence on the growth of plants, animals andmicroorganisms and it is known that compounds which inhibit theplant-derived PPO activity have generally the herbicidal activity. Thus,in order to effectively develop PPO inhibiting-type herbicides, therehas been a need for a method for evaluating the ability of a compound toinhibit the PPO activity with simplicity.

SUMMARY OF THE INVENTION

Under the aforementioned circumstances, the present inventors studiedhard, which resulted in completion of the present invention.

The present invention provides:

1. A method for evaluating the ability of a compound to inhibit theprotoporphyrinogen oxidase activity, which comprises the steps of:

(1) culturing a transformant expressing a protoporphyrinogen oxidasegene present in a DNA fragment in a medium containing substantially noprotoheme compounds in each comparative system of the presence andabsence of a test compound to measure a growth rate of the transformantunder each condition, said transformant being resulted from a host celldeficient in the growing ability based on the protoporphyrinogen oxidaseactivity transformed with the DNA fragment in which a promoterfunctionable in the host cell and a protoporphyrinogen oxidase gene areoperatively linked, and

(2) determining the ability of the compound to inhibit theprotoporphyrinogen oxidase activity by comparing the growth rates(hereinafter refered to as “the present method ”).

2. A method for evaluating the ability of a compound to inhibit theprotoporphyrinogen oxidase activity, which comprises the steps of:

(1) culturing a transformant expressing a protoporphyrinogen oxidasegene present in a DNA fragment in a medium containing substantially noprotoheme compounds in each comparative system of the presence andabsence of a test compound to measure a growth rate of the transformantunder each condition, said transformant being resulted from a host celldeficient in the growing ability based on the protoporphyrinogen oxidaseactivity transformed with the DNA fragment in which a promoterfunctionable in the host cell, a protoporphyrinogen oxidase gene and aterminator functionable in the host cell are operatively linked, and

(2) determining the ability of the compound to inhibit theprotoporphyrinogen oxidase activity by comparing the growth rates.

3. A method for evaluating the ability of a compound to inhibit theprotoporphyrinogen oxidase activity, which comprises the steps of:

(1) culturing a transformant expressing a protoporphyrinogen oxidasegene present in the following (a) DNA fragment in a medium containingsubstantially no protoheme compounds in each comparative system of thepresence and absence of a test compound to measure a growth rate of thetransformant under each condition, said transformant being resulted froma host cell deficient in the growing ability based on protoporphyrinogenoxidase activity transformed with

(a) the DNA fragment in which a promoter functionable in the host celland controllable in its transcriptional activity, and aprotoporphyrinogen oxidase gene are operatively linked, and

(b) a DNA fragment in which a gene being capable of controlling thetranscriptional activity of the promoter in the above DNA fragment and apromoter having the transcriptional activity not controllable by thegene and functionable in the host cell are operatively linked, and

(2) determining the ability of the compound to inhibit theprotoporphyrinogen oxidase activity by comparing the growth rates.

4. A method for evaluating the ability of a compound to inhibit theprotoporphyrinogen oxidase activity, which comprises the steps of:

(1) culturing a transformant expressing a protoporphyrinogen oxidasegene present in the following (a) DNA fragment in a medium containingsubstantially no protoheme compounds in each comparative system of thepresence and absence of a test compound to measure a growth rate of thetransformant under each condition, said transformant being resulted froma host cell deficient in the growing ability based on theprotoporphyrinogen oxidase activity transformed with

(a) the DNA fragment in which a promoter functionable in the host celland controllable in its transcriptional activity, a protoporphyrinogenoxidase gene and a terminator functionable in the host cell areoperatively linked, and

(b) a DNA fragment in which a gene being capable of controlling thetranscriptional activity of the promoter in the above DNA fragment, apromoter having the transcriptional activity not controllable by thegene and functionable in the host cell, and a terminator functionable inthe host cell are operatively linked, and

(2) determining the ability of the compound to inhibit theprotoporphyrinogen oxidase activity by comparing the growth rates.

5. The method according to 1 or 3, which is characterized in that theprotoporphyrinogen oxidase gene is a protoporphyrinogen oxidase genederived from an animal or a plant.

6. The method according to 1 or 3, which is characterized in that theprotoporphyrinogen oxidase gene is a protoporphyrinogen oxidase genederived from a fat or Chlamydomonas reinhardtii.

7. The method according to 1 or 3, which is characterized in that thehost cell is a microorganism.

8. A rat-derived gene encoding a protein having the protoporphyrinogenoxidase activity.

9. A protoporphyrinogen oxidase gene encoding a protein having the aminoacid sequence shown by SEQ ID: No.1.

10. A gene encoding a protein having the protoporphyrinogen oxidaseactivity and having the amino acid sequence in which one or severalamino acids are deleted, substituted, modified or added in the aminoacid sequence shown by SEQ ID: No.1.

11. A protoporphyrinogen oxidase gene having the nucleotide sequenceencoding the amino acid sequence shown by SEQ ID: No.1.

12. A protoporphyrinogen oxidase gene having the nucleotide sequenceshown by SEQ ID: No.2.

13. A DNA fragment having a partial nucleotide sequence of theprotoporphyrinogen oxidase gene of any one of 8 to 12.

14. A vector which comprises the protoporphyrinogen oxidase gene of anyone of 8 to 12.

15. A transformant which is characterized in that the vector of 14 isintroduced in to a host cell.

16. The transformant according to 15, wherein the host cell is amicroorganism.

17. The transformant according to 15, wherein the host cell is a plant.

18. A Chlamydomonas reinhardtii-derived gene encoding a protein havingthe protoporphyrinogen oxidase activity.

19. A protoporphyrinogen oxidase gene encoding a protein having theamino acid sequence shown by SEQ ID: No.9.

20. A gene encoding a protein having the protoporphyrinogen oxidaseactivity and having the amino acid sequence in which one or severalamino acids are deleted, substituted, modified or added in the aminoacid sequence shown by SEQ ID: No.9.

21. A protoporphyrinogen oxidase gene having the nucleotide sequenceencoding the amino acid sequence shown by SEQ ID: No.9.

22. A protoporphyrinogen oxidase gene having the nucleotide sequenceshown by SEQ ID: No. 10.

23. A DNA fragment having a partial nucleotide sequence of theprotoporphyrinogen oxidase gene of any one of 18 to 22.

24. A vector which comprises the protoporphyrinogen oxidase gene of anyone of 18 to 22.

25. A transformant which is characterized in that the vector of 24 isintroduced in a host cell.

26. The transformant according to 25, wherein the host cell is amicroorganism.

27. The transformant according to 25, wherein the host cell is a plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a method for constructing a vector containing a full lengthrat-derived protoporphyrinogen oxidase cDNA.

[PPO fragment 1] shows a DNA fragment obtained by a polymerase chainreaction of Example 2, [PPO fragment 2] shows a DNA fragment harbored bya clone obtained in Example 1, [PPO] shows a rat-derivedprotoporphyrinogen oxidase cDNA, and other symbols show a restrictionenzyme recognition site.

FIG. 2 shows a method for constructing a protoporphyrinogen expressionvector which is constructed for expressing a rat-derivedprotoporphyrinogen oxidase by introducing it into Escherichia coli.

[PPO] shows a rat-derived protoporphyrinogen oxidase cDNA, [lacZ] showsa beta-galactosidase gene, and other symbols show a restriction enzymerecognition site.

FIG. 3 shows a method for constructing a lac repressor expression vectorwhich is constructed for expressing a lac repressor by introducing itinto Escherichia coli.

FIG. 4 shows the structure of a compound which was subjected to a teston the ability of a compound to inhibit the rat-derivedprotoporphyrinogen oxidase activity.

FIG. 5 shows a method for constructing a protoporphyrinogen oxidaseexpression vector for direct introduction which is constructed forexpressing a rat-derived protoporphyrinogen oxidase by introducing itinto a plant cell.

[35S] shows the cauliflower mosaic virus-derived 35S promoter, [NOS]shows the Agrobacterium-derived nopaline synthase terminator, [PPO]shows a rat-derived protoporphyrinogen oxidase cDNA, and other symbolsshow a restriction enzyme recognition site.

FIG. 6 shows a method for constructing a protoporphyrinogen oxidaseexpression vector for indirect introduction which is constructed for arat-derived protoporphyrinogen oxidase by introducing it into a plantcell.

[35S] shows the cauliflower mosaic virus-derived 35S promoter, [NOS]shows the Agrobacterium-derived nopaline synthase terminator, [PPO]shows a rat-derived protoporphyrinogen oxidase cDNA, [NTPII] shows akanamycin-resistant gene, [NOSp] shows the Agrobacterium-derivednopaline synthase promoter, [LB] and [RB] show the AgrobacteriumT-DNA-derived left border nucleotide sequence and right bordernucleotide sequence, respectively, and other symbols show a restrictionenzyme recognition site.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will be described in detail.

First, the present method will be described.

A PPO gene used in the present method may be any ones encoding a proteinhaving the PPO activity upon expression in the host cell and, forexample, may be selected from PPO genes derived from plants and animals.More particularly, there are PPO genes derived from Dicotyledonousplants such as Arabidopsis, soybean, oil seed rape, sugar beat, potatoand tobacco, Monocotyledonous plants such as corn, rice, wheat, barley,oat, rye, sugar cane and sorghum, algae such as Chlamydomonasreinhardtii and Chlorella, mammals such as mouse, rat and human, fishessuch as trout, bluegill, carp, cyprinodont, guppy, zebra fish andfathead minnow, insects such as fly, mosquito, cockroach, greasy grind,dragonfly and silkworm moth.

As used herein, “a promoter functionable in a host cell” used in thepresent method is a DNA fragment having the transcriptional activity ina host cell being transformed and includes, for example, Escherichiacoli lactose operon promoter (lacP), tryptophan operon promoter (trpP),arginine operon promoter (argP), galactose operon promoter (gaiP), tacpromoter, T7 promoter, T3 promoter, λ phage promoter (λ-pL, λ-pR), yeastalcohol dehydrogenase gene (ADH) promoter, adenovirus major late (Ad.ML)promoter, SV40 early promoter, baculovirus promoter, nopaline synthasegene (NOS) promoter, octopine synthase gene (OCT) promoter, cauliflowermosaic virus (CaMV)-derived 19S and 35S promoters, phenylalanine ammonialyase (PAL) gene promoter, chalcone synthase (CHS) gene promoter and thelike.

In addition, “a terminator functionable in a host cell” used ifnecessary includes any DNA fragments having the transcriptionterminating activity in a host cell being transformed. Examples thereofinclude Escherichia coli lactose operon terminator, arginine operonterminator, galactose operon terminator, yeast HIS terminator, ADH1terminator, SV40 early splicing region, nopaline synthase gene (NOS)terminator, garlic virus GV 1 and GV2 terminators and the like.

As used herein, “a DNA fragment are operatively linked” means a DNAfragment in which these promoter, and, if necessary, these terminator,are linked so that the above PPO gene is expressed under the control ofthe above promoter, and, if necessary, the above terminator, in a hostcell into which they are to be introduced (hereinafter referred to as“expression DNA fragment”).

The above expression DNA fragment is introduced into a host celldeficient in the growing ability based on the PPO activity to obtain atransformant expressing the introduced PPO gene.

A host cell being transformed, that is, deficient in the growing abilitybased on the PPO activity may be any host cells deficient in the PPOactivity necessary for growth and, from a viewpoint of simple culturing,microorganisms are preferable. Examples of microorganisms deficient inthe PPO activity necessary for growth include PPO gene (hemGlocus)-deficient mutant Escherichia coli strain VSR 751 described in K.Miyamoto, K. Nakahigashi, K. Nishimura, T. Nakayashiki and H. Inokuchi,(1991) Journal of Molecular Biology, vol.219, pp393-398 and K.Nishimura, T. Nakayashiki and H. Inokuchi, (1993) Gene, vol.133,pp109-113, PPO gene (hemG locus)-deficient mutant Escherichia colistrain BT3 described in F. Yamamoto, H. Inokuchi and H. Ozeki, (1988)Jpn. J. Genet., vol.63, pp237-249, PPO gene (hem14-1 locus)-deficientmutant yeast strain hem14-1 described in J. M. Gamadro, D. Urban-Grimaland P. Labbe (1982) Biochem. Biophys. Res. Commun., vol.106, pp724-730,and the like.

The above expression DNA fragment incorporated into a vector may beintroduced into a host cell. The vector containing the expression DNAfragment may be introduced into a host cell according to theconventional genetic engineering method. For example, when the host cellis a microorganism, the vector may be introduced into a cell of themicroorganism with a calcium chloride method, an electroporation method(Methods in Electroporation: Gene Pulser/E. coli Pulser System, Bio-RadLaboratories, 1993) and the like. In addition, when the host cell is aplant, the vector may be introduced into a cell of the plant with anAgrobacterium infection method (JP-B-2-58917 (examined) andJP-A-60-70080 (laid-opened)), an electroporation into a protoplastmethod (JP-A-60-251887 (laid-opened) and JP-A-5-68575 (laid-opened)), aparticle gun method (JP-A-5-508316 (laid-opened) and JP-A-63-258525(laid-opened)) and the like. A transformant expressing the introducedPPO gene may be obtained by culturing the host cell with the introducedvector in a medium containing a growth inhibitor corresponding to aselectable marker lucus-linked on the vector and a medium containingsubstantially no protoheme compounds, and isolating a clone which cangrow in the medium.

The transformant thus obtained is first cultured in a medium containingsubstantially no protoheme compounds in each comparative system of thepresence at a variety of concentrations and absence of a test compoundto measure a growth rate of the transformant under each condition. Next,the PPO activity inhibiting ability of the test compound to inhibit thePPO activity is determined by comparing the growth rates based on theeach comparative system of contact and non-contact of the transformantand the test compound. The comparison of the growth rates is performed,for example, by obtaining the growth inhibiting rate by calculating aratio of the growth rate in a system of the presence of the testcompound relative to the growth rate in a system of the absence of thetest compound, and comparing the growth inhibiting rates regarding aplurality of test compounds and, based on the results thereof, thehigher or lower ability for inhibiting PPO activity may be determined.More particularly, for example, a concentration of a test compound atwhich the growth is inhibited by 50% is obtained from the growth rate ofthe transformant in the system of the addition of a compound (that is,of the presence of the test compound) under the conditions that thegrowth rate of the transformant is 100% in the system of no addition ofa compound (that is, of the absence of the test compound). The testcompound having the lower concentration may be determined to be higherin the ability for inhibiting PPO activity than the test compound havingthe higher concentration. Alternatively, a compound known to inhibit thePPO activity (positive control) is simultaneously subjected to anexperiment as one of test compounds, which may serve as an index for thegrowth inhibiting rate of each test compound (for example, a compoundconcentration at which the growth is inhibited by 50%).

As used herein, “a medium containing substantially no protohemecompounds” means a medium not containing any protpheme compounds or amedium which may contain protoheme compounds at such an amount that ahost cell deficient in the growing ability based on the PPO activity cannot recover the growing ability to the same level as that of thecorresponding non-deficient host cell (that is, a host cell notdeficient in the growing ability based on the PPO activity) regardlessof the presence of protoheme compounds in the medium. The examplesthereof include an artificial medium not containing any protohemecompounds such as protoporphyrin, protohemin and the like at all, amedium containing natural extracts (for example, yeast extract, Maltextract and the like), and the like. From a viewpoint of the precisionof the present method, it is preferable that an amount of the protohemecompounds present in a medium is zero or as low as possible. Inaddition, protoheme compounds include protoheme and its derivatives andthese compounds mean a compound having the action of recovering thegrowing ability of a host cell deficient in the growing ability based onthe PPO activity.

In order to confirm that the growth inhibition by a test compound is dueto the inhibition of PPO activity, it can be easily determined byperforming the culturing of the above transformant in a mediumcontaining substantially protoheme compounds such as protoporphyrin,protohemin and the like, and confirming the growth of the transformant.In addition, if needed, the growth rate of the transformant may beconveniently adjusted by the relationship between an expressed amount ofPPO gene and a concentration of a test compound.

In the present method, the expressed amount of PPO gene in thetransformant may vary by using “a promoter functionable in a host celland controllable in its transcriptional activity” as a promoter for theaforementioned expression DNA fragment, and introducing into the hostcell “a DNA fragment in which a gene capable of controlling thetranscriptional activity of a promoter in the expression DNA fragmentand a promoter having the transcriptional activity not-controllable bythe gene and functionable in the host cell, and an optionally usedterminator functionable in the host cell are operatively linked”(hereinafter referred to as “regulatory DNA fragment”). For exemple,when evaluation of a test compound having the lower water-solubility andthat of a small amount of a test compound are performed, by adjusting anexpressed amount of PPO gene in the transformant to be small, evaluationmay be performed with the higher precision.

On the other hand, when evaluation of a test compound having the higherability for inhibiting PPO activity are performed, by adjusting anexpressed amount of PPO gene in the transformant to be increased,evaluation may be performed with the higher precision.

As used herein, “a gene capable of controlling the transcriptionalactivity of a promoter in the expression DNA fragment” (hereinafterreferred to as “a regulatory gene”) means a gene having the function ofinhibiting or promoting the transcriptional activity of “a promoterfunctionable in the host cell and controllable in its transcriptionalactivity” as a promoter for the expression DNA fragment. When anexpressed amount of PPO gene is to be decreased, a gene which inhibitsthe transcriptional activity of a promoter in the expression DNAfragment may be used, while when an expressed amount of PPO gene is tobe increased, a gene which promotes the transcriptional activity of thepromoter may be used. More particularly, examples of a combination forinhibiting the transcriptional activity include a combination ofEscherichia coli lactose operon promoter and lacI, that of arginineoperon promoter and argR, that of galactose operon promoter and GaIR andthe like. On the other hand, examples of a combination for promoting thetranscriptional activity include a combination of Escherichia colilactose operon promoter and crp, that of Klebsiella pneumoniae maltoseregulon and malT and the like.

“A promoter having the transcriptional activity not-controllable by theregulatory gene and functionable in a host cell”, that is, a promoterfor the regulatory DNA fragment may be any promoters having no influenceby a gene which they themselves control. For example, in a case of apromoer to be combined with a repressor gene for the above lactoseoperon, its own promoter, nopaline synthase enzyme gene (NOS) promoterand the like may be used.

The above expression DNA fragment and the regulatory DNA fragment may beintroduced into a host cell by incorporating into the same or differentvector and these DNA fragments may be incorporated into chromosome(s) ofthe host cell.

The present method may be utilized for screening medications having theability for inhibiting the malignant cell growth in addition toevaluating the potency of a phototrophic herbicide, screening a compoundhaving the hebicidal activity.

When the present method is utilized for evaluating the potency of aphototrophic herbicide or screening a compound having the herbicidalactivity, the present method is performed, for example, using PPO genesderived from higher plants such as Arabidopsis, PPO genes derived fromalgae such as Chlamydomonas reinhardtii. As a result, a compoundinhibiting the growth of the transformant into which a PPO gene derivedfrom higher plants or algae is introduced is potentially useful as anactive ingredient of a herbicide.

Next, a gene which may be used for the present method will be describedin detail.

Examples of a rat-derived gene encoding a protein having the PPOactivity (hereinafter referred to as “the present rat PPO gene”) are (1)a rat-derived gene having about 1.64 kbp of length, for example, a PPOgene encoding a protein having the amino acid sequence shown by SEQ ID:No. 1, (2) a gene encoding a protein having the PPO activity and havingthe amino acid sequence in which one or several amino acids are deleted,substituted, modified or added in the amino acid sequence shown in SEQID: No.1. More particularly, for example, mention be made of a PPO genehaving the nucleotide sequence shown by SEQ ID: No.2.

Examples of a Chlamydomonas reinhardtii-derived gene encoding a proteinhaving the PPO activity (hereinafter referred to as “the presentChlamydomonas reinhardtii PPO gene”) are (1) a Chlamydomonasreinhardtii-derived gene having about 2 kbp of length, for example, aPPO gene encoding a protein having the amino acid sequence shown by SEQID: No.9, (2) a gene encoding a protein having the PPO activity andhaving the amino acid sequence in which one or several amino acids aredeleted, substituted, modified or added in the amino acid sequence shownin SEQ ID: No.9. More particularly, for example, mention be made of aPPO gene having the nucleotide sequence shown by SEQ ID: No.10.

The present rat PPO gene is obtained, for example, according to thefollowing method.

RNA is extracted from rat tissues such as liver, kidney and the likeaccording to a method described in Labomanual Genetic Engineering,Suppl. Edition, edited by Matsumura, published by Maruzen K.K., pp76-77,1990. The extracting procedures may be carried out utilizing acommercially available RNA extracting kit, for example, ISOGEN (NipponGene).

The resultant RNA is subjected to the manipulation according to theattached manual using a commercially available poly(A) RNA fractionatingkit such as BIOMAG mRNA Purification kit (Perceptive Biosystems) toprepare poly(A) RNA. The resulting poly(A) RNA is subjected to themanipulation according to a method described in Cloning and Sequence:Plant Biotechnology Experimental Manual, edited by Watanabe and Sugiura,published by Nosonbunka Company, pp74-103 (1989) to make cDNA library.This cDNA library making manipulation may utilize a commerciallyavailable cDNA library making kit such as ZAP-cDNA Gigapack Cloning Kit(STRATAGENE).

From thus made cDNA library or a commercially available cDNA librarysuch as a rat liver-derived cDNA liver manufactured by STRATAGENE, DNAmay be prepared by a method described in Molecular Cloning 2nd edition(authors: J. Sambrook, E. F. Frisch, and T. Maniatis; Cold Spring HarborLaboratory Press, 1989) 2.60-2.81.

By using thus prepared DNA as a template, a polymerase chain reactionmay be performed using an oligonucleotide having the nucleotide sequenceshown by SEQ ID: No.3 and that having the nucleotide sequence shown inSEQ ID: No.4 as a primer to amplify a DNA fragment having a partialnucleotide sequence of PPO gene. This amplified DNA fragment having apartial nucleotide sequence of a PPO gene may be cloned into a plasmidusing the conventional method described in Labomanual GeneticEngineering, Suppl. Edition, edited by Matsumura, published by MaruzenK.K., pp117-120 (1990) and a commercially available DNA cloning kit suchas TA cloning kit (manufactured by Invitrogen) to serve for determiningnucleotide sequence. Determination of the nucleotide sequence may beperformed by a dideoxy method described, for example, in MolecularCloning 2nd edition (authors: J. Sambrook, E. F. Frisch and T. Maniatis;Cold Spring Harbor Laboratory Press), 13.42-13.74 (1989). Determinationof the nucleotide sequence according to dideoxy method may be performedutilizing a sequencing kit using fluorescently-labeled dideoxynucleotide(more particularly, for example, Dye terminator cycle sequencing kit(manufactured by PE Applied Biosytems) and using a commerciallyavailable autosequencer such as DNA Sequencer 373S (manufactured by PEApplied Biosytems).

Thus, the DNA fragment having a partial nucleotide sequence of a rat PPOgene (hereinafter referred to as “the present rat PPO DNA fragment”) canbe obtained. One example of the present rat PPO DNA fragment is a genefragment having the nucleotide sequence shown in nucleotide Nos. 639 to1333 shown by SEQ ID: No.2. The present rat PPO DNA fragment may beutilized in making a PPO gene-specific primer used in a polymerase chainreaction described below or used as a probe for detecting a PPO gene ina hybridization method described below.

Based on the present rat PPO DNA fragment, the present rat PPO genehaving entire nucleotide sequence encoding PPO may be obtained accordingto (1) polymerase chain reaction method or (2) hybridization method.

In order to obtain a PPO gene by utilizing a polymerase chain reaction,a primer having about 15 bp to about 40 bp nucleotide sequence among thenucleotide sequence of the present rat PPO DNA fragment (primer foramplifying 3′ downstream region) and about 15 bp to about 40 bpnucleotide sequence among the nucleotide sequence complementary to thebase sequence of the present rat PPO DNA fragment (primer for amplifying5′ upstream region) are first prepared. Examples of the primer foramplifying 3′ downstream region are a gene fragment having thenucleotide sequence shown in base nucleotide Nos.1175 to 1198 by SEQ ID:No.2 and the like, and examples of the primer for amplifying 5′ upstreamregion are a gene fragment having the nucleotide sequence complementaryto the nucleotide sequence shown in nucleotide Nos.776 to 799 by SEQ ID:No.2 and the like.

Then, a polymerase chain reaction is performed using as a template a DNAfragment having an adapter DNA added at an end of a rat-derived DNA or aDNA of a vector having rat-derived DNA fragment inserted therein andusing a combination of a primer having a partial nucleotide sequence ofan adapter DNA or a primer having a partial nucleotide sequence of avector DNA and a primer for amplifying 5′ upstream region and acombination of a primer having a partial nucleotide sequence of anadapter DNA or a primer having a partial nucleotide sequence of a vectorDNA and a primer for amplifying 3′ downstream region to amplify a DNAfragment containing 5′ upstream nucleotide sequence of the present ratPPO DNA fragment and a DNA fragment having 3′ downstream nucleotidesequence of the present rat PPO DNA fragment. The DNA fragment having anadapter DNA added at an end of a rat-derived DNA which is used as atemplate may be prepared by adding as an adapter DNA a polymer of anucleotide such as cytosine to a rat-derived cDNA prepared above withterminal deoxynucleotidyl transferase or adding a commercially availableadapter of a kit for RACE (rapid amplification of cDNA ends) reactionsuch as an adapter attached to Marathon cDNA amplification kitmanufactured by Clontech to a rat-derived cDNA. In addition, one exampleof the primer having a partial nucleotide sequence of an adapter DNA isa primer specific for an adapter attached to a commercially availableRACE (rapid amplification of cDNA ends) reaction kit (for example, AP-1primer and AP-2 primer attached to Marathon cDNA amplification kitmanufactured by Clontech). As the primer having a partial sequence of avector DNA, when a vector is derived from λ phage, primers specific foran arm region of λ phage such as λ gtII reverse primer and λ gtIIforward primer may be used.

Two kinds of thus amplified DNA fragments may be cloned into a plasmidby using the conventional method described in Labomanual GeneticEngineering, Suppl. Edition, edited by Matsumura, published by MaruzenK.K., pp117-120 (1990) or a commercially available DNA cloning kit suchas TA cloning kit (manufactured by Invitrogen) to serve fordetermination of the nucleotide sequence. Determination of thenucleotide sequence may be performed by a dideoxy method described inMolecular Cloning 2nd edition (authors: J. Sambrook, E. F. Frisch and T.Maniatis; Cold Spring Harbor Laboratory Press), 13.42-13.74 (1989).Determination of the nucleotide sequence by a dideoxy method may beperformed by utilizing a sequencing kit using a fluorescently labeleddideoxynucleotide such as Dye terminator cycle sequencing kit(manufactured by PE Applied Biosystems) and using a commerciallyavailable autosequencer such as DNA Sequencer 373S (manufactured by PEApplied Biosystems). The regions between the thus determined nucleotidesequence and the nucleotide sequence of the present rat PPO DNA fragmentwhich was referenced upon manufacturing the above primer are overlaid toligate these nucleotide sequences to make one base sequence. Whether ornot an entire region of open reading frame encoding PPO is contained inthe thus made nucleotide sequence may be studied by investigating openreading frame using a commercially available gene analyzing softwaresuch as GENETYX (manufactured by SDC). When a 1.6 kbp or more entireopen reading frame is not contained in the determined nucleotidesequence and translation initiation codon or translation terminationcodon are not contained therein, it is determined that the wholenucleotide sequence of the full length PPO gene is not contained in thenucleotide sequence, and steps for obtaining a gene fragment by apolymerase chain reaction is repeated until PPO gene having the wholenucleotide sequence encoding PPO is obtained. In addition, when openreading frame which is found in the nucleotide sequence determined asdescribed above is missing in either in 5′ upstream or 3′ downstreamside of the determined nucleotide sequence, that is, when a translationinitiation codon or translation termination codon is not contained inthe open reading frame, steps for obtaining a gene fragment by thepolymerase chain reaction same as above may be repeated for obtaining aDNA fragment containing the nucleotide sequence on the missing's side.

Based on the information of the nucleotide sequence thus determined, aprimer containing the nucleotide sequence near translation initiationcodon in the nucleotide sequence of a PPO gene (N-terminal primer) and aprimer containing the nucleotide sequence complementary to thenucleotide sequence near translation termination codon (C-terminalprimer) are made, and a polymerase chain reaction may be performed usinga rat-derived DNA as a template and using the above both terminalprimers to amplify the present rat PPO gene having the entire nucleotidesequence encoding PPO, which may be cloned. Examples of the N-terminalprimer include a primer having the nucleotide sequence shown by SEQ ID:No. 5 and a primer having the nucleotide sequence shown by SEQ ID: No.7, and examples of the C-terminal primer include a primer having thenucleotide sequence shown by SEQ ID: No. 6 and a primer having thenucleotide sequence shown by SEQ ID: No. 8. Alternatively, the DNAfragments obtained by the above steps for obtaining the gene fragmentmay be ligated with an restriction enzyme recognition site present inthe overlapping regions to obtain the present rat PPO gene having theentire nucleotide sequence encoding PPO.

(2) In order to obtain the present rat PPO gene by utilizing ahybridization method, a DNA fragment may be identified by hybridizing aprobe of the present rat PPO DNA fragment to a rat-derived DNA and theidentified DNA fragment may be isolated.

A probe used for a hybridization method may be prepared by labeling thepresent rat PPO DNA fragment obtained as described above with a labelingcompound by the conventional method described in Molecular Cloning 2ndedition (authors: J. Sambrook, E. F. Frisch and T. Maniatis; Cold SpringHarbor Laboratory Press),10.6-10.26 (1989). A labeling compound such asa compound containing a radioactive element (hereinafter referred to as“RI”) and a fluorescent reagent and the like may be used. Labeling witha compound containing RI may be performed using [α-³²P] dCTP(manufactured by Amersham) and a random prime labeling kit (manufacturedby Boehringer Mannheim) according to the protocol attached to the kit.In addition, non-RI labeling may be performed using DIG-High Prime DNALabeling and Detection Starter KitI (Boehringer Mannheim) and attachedreagents according to the attached protocol.

As a rat-derived DNA for hybridizing with a probe, a rat cDNA libraryand the like may be used which is obtained by ligating a cDNA derivedfrom a rat to a vector with a suitable adaptor, derived from λ phage andthe like and packaging the cDNA into phage particles. Such the genelibrary may be prepared according to a method, for example, described inCloning and Sequencing: Plant Biotechnology Experimental Mannual,editted by Watanabe and Sugiura, published by Nosonbunka, pp74-103(1989). Alternatively, the rat gene libraries sold by Clontech,STRATAGENE and the like may be used.

In order to isolate the present gene, for example, colony hybridization,plaque hybridization and the like to the aforementioned rat gene librarymay be performed using the probe prepared by the aforementioned method.These may be performed according to a method described in RecombinantDNA Experiment (edited by J. R. Dillon, A. Nasim and E. R. Nestnann,published by Tokyo kagakudozin), pp98-101 (1987) or the conventionalmethod described in Molecular Cloning 2nd edition (authors: J. Sambrook,E. F. Frisch and T. Maniatis; Cold Spring Harbor Laboratory Press),pp1.90-1.104 or 2.108-2.117 (1989). Determination of the nucleotidesequence of the DNA fragment incorporated in the resultant clone may beperformed by a dideoxy method described in Molecular Cloning 2nd edition(authors: J. Sambrook, E. F. Frisch and T. Maniatis, Cold Spring HarborLaboratory Press), pp13.42-13.74 (1989). The determination of thenucleotide sequence by a dideoxy method may be performed by utilizing asequencing kit using fluorescently labeled dideoxynucleotide such as Dyeterminator cycle sequencing kit (manufactured by PE Applied Biosystems)and using a commercially available autosequencer such as DNA Sequencer373S (manufactured by PE Applied Biosystems). The determined nucleotidesequence may be analyzed using a commercially available gene analyzingsoftware such as GENETYX (SDC) to reveal the entire nucleotide sequenceof the present rat PPO gene.

Next, the present Chlamydomonas reinhardtii-derived PPO gene is obtainedby the following method. The fundamental manipulation may be performedaccording to the aforementioned description for obtaining the presentrat PPO gene.

Chlamydomonas reinhardtii is cultured to collect cells and RNA isextracted from the collected cells. A cDNA library is made from theresultant RNA according to a method for obtaining the present rat PPOgene to prepare its DNA.

By using the DNA thus prepared as a template, a polymerase chainreaction may be performed using as a primer an oligonucleotide havingthe nucleotide sequence shown by SEQ ID: No.11 and that having thenucleotide sequence shown by SEQ ID: No. 12 to amplify a DNA fragmenthaving a Chlamydomonas reinhardtii PPO gene. The amplified DNA fragmenthaving the nucleotide sequence of PPO gene may be cloned into a plasmidto serve for determination of the nucleotide sequence.

Thus obtained cDNA of the present rat PPO gene or the present rat PPODNA fragment, or the cDNA of the present Chlamydomonas reinhardtii PPOgene may be used to obtain a genome DNA clone of the present rat PPOgene or the present Chlamydomonas reinhardtii PPO gene, the nucleotidesequence of which may be determined. First, in the case of the genomeDNA clone of the present rat PPO gene, for example, rat tissues such asliver, kidney and the like are taken and a genome DNA is extracted fromthe taken tissues by manipulations according to a method described inLabomanual Genetic Engineering, Suppl. Edition, edited by Matsumura,published by Maruzen K.K., pp76-77 (1990). On the other hand, in thecase of a genome clone of the present Chlamydomonas reinhardtii PPOgene, for example, Chlamydomonas reinhardtii is cultured to collectcells and a genome DNA is extracted from the collected cells accordingto the similar procedures. The extracting procedures may be performedusing a commercially available DNA extracting kit such as ISOTISSUE(Nippon Gene) according to the protocol attached to the kit to obtain agenome DNA. After the genome DNA is cut with a suitable restrictionenzyme, the resultant genome DNA fragment is fractionated according to aNaCl density gradient centrifugation method described in Cloning andSequencing: Plant Biotechnology Experimental Manual, editted by Watanabeand Sugiura, published by Nosonbunka, pp276-279 (1989). In general,conveniently, a fraction containing a genome DNA fragment having a sizesuitable for incorporating into a vector, particularly, a fractioncontaining 9 kbp to 23 kbp genome DNA fragment when a phage vector isused, a fraction containing 30 kbp to 42 kbp genome DNA fragment when acosmid vector is used is selected from the fractionated respectivegenome DNA fragments. A genome library is made using the resultantgenome DNA fragments and performing the procedures according to a methoddescribed in Cloning and Sequencing: Plant Biotechnology ExperimentalManual, edited by Watanabe and Sugiura, published by Nosonbunka,pp96-103 and 280-284 (1989) or using a commercially available DNAlibrary making kit such as Lambda EMBL3/Gigapack cloning kit(STRATAGENE) according to the attached protocol. The genome DNA librarythus made, or a commercially available genome library such as rat genomelibrary manufactured by STRATAGENE is screened by hybridization using asa probe a cDNA of the present rat PPO gene or the present rat PPO DNAfragment, or a cDNA of the present Chlamydomonas reinhardtii PPO geneaccording to a method described in Cloning and Sequence: PlantBiotechnology Experimental Manual, edited by Watanabe and Sugiura,published by Nosonbunka, pp106-147 (1989) to obtain a genome DNA clonehaving the nucleotide sequence of the present rat PPO gene, or thepresent Chlamydomonas reinhardtii PPO gene. The resultant genome DNAclone may be subcloned into a suitable vector for analyzing thenucleotide sequence such as a plasmid and the like according to a methoddescribed in Cloning and Sequence: Plant Biotechnology ExperimentalMannual, edited by Watanabe and Sugiura, published by Nosonbunka,pp152-174 (1989), and sequenced by a dideoxy method and the likeaccording to a primer extension method described in Molecular Cloning2nd edition (authors; J. Sambrook, E. F. Frisch and T. Maniatis; ColdSpring Harbor Laboratory), pp13.15 (1989). Determination of thenucleotide sequence by dideoxy method may be performed using acommercially available kit such as Dye terminator cycle sequencing kitmanufactured by PE Applied Biosystems and using a DNA sequencer such asModel 373S of PE Applied Biosystems.

Further, by a primer extension method described in Bina-Stem Met et al.,(1979) Proc. Natl. Acad. Sci.U.S.A.,vol.76, pp731 and Sollner-Webb andR. H. Reeder, (1979) Cell, vol.18, pp485 or a S1 mapping methoddescribed in A. J. Berk and P. A. Sharp, (1978) Proc. Natl. Acad. Sci.U.S.A., vol.75, pp1274 and the like, each transcription initial point ofthe genome DNA of the present rat PPO gene or the present Chlamydomonasreinhardtii PPO gene may be decided. At about 1 kb to about 10 kbupstream from this transcription initiation point, there is a promotersequence responsible for controlling the gene expression.

In addition, the present rat PPO gene and the present Chlamydomonasreinhardtii PPO gene can be utilized as a means for obtaining orgenerating a gene resistance to a PPO activity inhibiting agent known asa phototrophic herbicide. For example, it becomes possible to generate anew herbicide-resistant gene by introducing into a PPO gene a mutationwhich gives a change to the amino acid sequence of PPO encoded by thepresent rat PPO gene or the present Chlamydomonas reinhardtii PPO geneand screening a gene encoding the amino acid sequence of PPO showingphototrophic herbicide-resistance. More particularly, it becomespossible to generate a new phototrophic herbicide-resistant gene byinducing a random mutation in the nucleotide sequence of the present ratPPO gene or the present Chlamydomonas reinhardtii PPO gene to introducea mutation into the amino acid sequence according to a method describedin A. Greener, M. Callahan, Strategies, 1994, Vol.7, pp32 to 34, and thelike. In addition, it also becomes possible to generate a newphototrophic herbicide-resistant gene by introducing a site-specificmutation into the nucleotide sequence of the present rat PPO gene or thepresent Chlamydomonas reinhardtii PPO gene to alter the amino acidsequence according to a gapped duplex method described in W. Kramer, etal., Nucleic Acids Research, 1984, vol.12, pp9441 or W. Kramer, H. J.Frits, Methods in Enzymology, 1987, vol.154, pp350, or a Kunkel methoddescribed in T. A. Kunkel, Proc. of Natl. Acad. Sci. U.S.A., 1985,vol.82, pp488 or T. A. Kunkel, et. al., Methods in Enzymology, 1987,vol. 154, pp367, and the like. Further, it also becomes possible togenerate a new phototrophic herbicide-resistant gene by making a geneencoding a chimera protein in which one or several partial amino acidsequences among the amino acid sequence of PPO encoded by the presentrat PPO gene or the present Chlamydomonas reinhardtii PPO gene aresubstituted with a part of the amino acid sequence of the presentChlamydomonas reinhardtii PPO gene, the present rat PPO gene or PPO genederived from other organisms. The effects of the thus made phototrophicherbicide-resistant gene (expresasion of herbicide resistance) can beeffectively confirmed by, for example, introducing a generatedherbicide-resistant gene into a microorganism defective in PPO or amicroorganism having PPO sensitive to a subject herbicide, selecting atransformed microorganism, and treating the transformed microorganismwith the subject herbicide to reselect a herbicide-resistant colony.

As a vector which comprises the present rat PPO gene or the presentChlamydomonas reinhardtii PPO gene, mention may be made of a plasmidmade by cloning into pCR2.1 (Invitrogen) a PPO gene having thenucleotide sequence encoding the amino acid sequence shown by SEQ ID:No.1 or No.9 and the plasmid has the such the characteristics that avector part is small and a copy number is large in Escherichia coli and,thus, is suitable for preparing DNA or analyzing the DNA structure.

A vector which can cause the present rat PPO gene or the presentChtamydomonas reinhardtii PPO gene to be expressed in a host cell can beconstructed by, for example, inserting into a vector a DNA fragment inwhich (1) a promoter functionable in a host cell, and (2) the presentrat PPO gene or the present Chlamydomonas reinhardtii PPO gene and, ifnecessary, (3) a terminator functionable in a host cell are operativelylinked in an operative manner in the above order. As used herein,“operatively” means that the present rat PPO gene or the presentChlamydomonas reinhardtii PPO gene are ligated to a promoter (and, ifnecessary, a terminator) so that they are expressed under the control ofthe promoter (and the terminator) upon introduction of the vector intothe host cell to transform the host cell.

As a “a promoter functionable in a host cell” is not limited tospecified ones but may be any promoters having the transcriptionalactivity in a host cell to be transformed. For example, mention may bemade of Escherichia coli lactose operon promoter, yeast alcoholdehydrogenase gene (ADH) promoter, adenovirus major late (Ad.ML)promoter, SV40 early promoter, baculovirus promoter, nopaline synthasegene (NOS) promoter, octopine synthase gene (OCT) promoter, cauliflowermosaic virus (CaMV)-derived 19S and 35S promoters, phenylalanine ammonialyase (PAL) gene promoter, chalcone synthase (CHS) gene promoter and thelike.

“A terminator functionable in a host cell” is not limited to specifiedones but may be any terminators having the transcription terminatingactivity in a host cell to be transformed. For example, mention may bemade of Escherichia coli lactose operon terminator, yeast HISterminator, ADH1 terminator, SV40 early splicing region, nopalinesynthase gene (NOS) terminator, garlic virus GV1, GV2 terminators andthe like.

A host cell can be transformed by introducing the above vector into thehost cell. For example, when a host cell is a microorganism, the abovevector can be introduced into a cell of the microorganism by the knownmeans such as a calcium chloride method, an electroporation method(Methods in Electroporation: Gene Pulser/E. coli Pulser System, Bio-RadLaboratories, 1993) and the like. On the other hand, when a host cell isa plant, a transformed plant cell can be obtained by introducing theabove present vector into a cell of the plant by the known means such asan Agrobacterium infection method (JP-B-2-58917 (examined) andJP-A-60-70080 (laid-opened)), an electroporation to a protoplast method(JP-A-60-251887 (laid-opened) and JP-A-5-68575 (laid-opened)), or aparticle gun method (JP-A-5-508316 (laid-opened) and JP-A-63-258525(laid-opened)) to select a cell into which the present rat PPO gene orthe present Chlamydomonas reinhardtii PPO gene is introduced. From theresultant transformed plant cell, a transformed plant body can beobtained by regenerating a transformed plant by a plant cell culturingmethod described in, for example, Plant Genetic Manipulation Manual: Amethod for generating A Transgenic Plant (author: Uchimiya, KodanshaScientific, 1990, pp.27 to 55).

Furthermore, PPO activity of a host cell or sensitivity of a host cellto the inhibitor for PPO activity may be altered by transforming a hostcell by introducing into the host cell a vector containing a DNAfragment in which (1) a promoter functionable in a host cell and (2) thepresent rat PPO gene or the present Chlamydomonas reinhardtii PPO geneand, if necessary, (3) a terminator functionable in a host cell usingthe present rat PPO gene or the present Chlamydomonas reinhardtii PPOgene.

As used herein, “a promoter functionable in a host cell” is not limitedto specific ones but may be any promoters having the transcriptionalactivity in a host cell to be transformed. For example, mention may bemade of Escherichia coli lactose operon promoter, yeast alcoholdehydrogenase gene (ADH) promoter, adenovirus major late (Ad.ML)promoter, SV40 early promoter, baculovirus promoter, nopaline synthasegene (NOS) promoter, octopine synthase gene (OCT) promoter, cauliflowermosaic virus (CaMV)-derived 19S and 35S promoters, phenylalanine ammonialyase (PAL) gene promoter, chalcone synthase (CHS) gene promoter and thelike.

“A terminator functionable in a host cell” is not limited to specifiedones but may be any terminators having the transcription terminatingactivity in a host cell to be transformed. For example, mention may bemade of Escherichia coli lactose operon terminator, yeast HISterminator, ADHI terminator, SV40 early splicing region, nopalinesynthase gene (NOS) terminator, garlic virus GV1, GV2 terminators andthe like.

By the above method for altering a host cell, for example, a resistanceto a phototrophic herbicide targeting PPO can be imparted to a plant.

Other PPO genes can be obtained and utilized by the aforementionedmethod for obtaining the present rat PPO gene. For example, in order toobtain a rabbit-derived PPO gene, a commercially available rabbit cDNAlibrary, primers, etc shown by SEQ ID: No.3 and No. 4 and the like maybe used.

EXAMPLES

The following Examples illustrate the present invention in detail but donot limit the present invention.

Example 1 Cloning of a Rat PPO DNA Fragment

An oligonucleotide having the nucleotide sequence shown by SEQ ID: No.3and that having the nucleotide sequence shown by SEQ ID: No.4 wereprepared. The oligonucleotides were synthesized using a DNA synthesizer(PE Applied Systems: Model 394 DNA/RNA Synthesizer), and using as asolvent for synthesis a solvent for Model 394 DNA/RNA Synthesizer (PEApplied Systems) and using as a DNA synthesis reagent phosphoamiditereagents (PE Applied Systems) corresponding to adenine, cytosine,guanine and thymine. The synthesized oligonucleotides were purified byan oligonucleotide purifying cartridge (PE Applied Systems: OPCcartridge) and dried under reduced pressure to prepare oligonucleotides.

A library comprising λ ZAPII vector having inserted cDNA derived from arat liver (manufactured by STRATAGENE) (hereinafter referred to as “ratcDNA library”) was spread over several plates in NZCYM agar medium toamplify according to a method described in Molecular Cloning 2^(nd)edition (authors: J. Sambrook, E. F. Frisch, T. Maniatis; Cold SpringHarbor Laboratory Press, 1989), 2.60-2.65 and phage particles wereeluted with an SM buffer from the agar medium per each plate(hereinafter referred to as “amplified library”). A DNA was extractedfrom the amplified library using a DNA extracting kit (Lambda-TRAP PLUS:manufactured by Clontech) to prepare a phage cloned DNA. A polymerasechain reaction was performed using this phage cloned DNA as a templateto amplify the DNA fragment. A reaction solution for polymerase chainreaction was prepared by taking 10 pmol of an oligonucleotide having thenucleotide sequence shown by SEQ ID: No. 3, 10 pmol of anoligonucleotide having the nucleotide sequence shown by SEQ ID: No. 4, 5μl 10 X PCR buffer (manufactured by TAKARA SHUZO CO., LTD), 0.25 μl TaqDNA polymerase (TaKaRa Taq manufactured by TAKARA SHUZO CO., LTD), each10 nmol of four kinds of nucleotides (dATP, dCTP, dGTP, dTTP:manufactured by TAKARA SHUZO CO., LTD), and long of the phage clone DNAin a 0.5 ml volume of a polymerase chain reaction tube and addingsterile distilled water to total 50 μl. Each step of polymerase chainreaction was carried out under the following conditions: The first cyclecomprising a denaturing step holding a temperature at 95° C. for 1minute, an annealing step holding a temperature at 55° C. for 2 minutes,and an extension step with a DNA polymerase holding a temperature at 72°C. for 3 minutes was performed once, and the second cycle comprising adenaturing step holding a temperature at 95° C. for 1 minute, anannealing step holding a temperature at 55° C. for 1.5 minutes, and anextentsion step with a polymerase holding a temperature at 72° C. for 2minutes was performed 34 times. After completion of the polymerase chainreaction, the reaction solution was analyzed on an agarose gelelectrophoresis to select an amplified library from which about 700 bpamplified DNA fragment is detected.

Next, Escherichia coli was infected with the selected amplified libraryand pBluescript vector containing a rat cDNA was prepared according tothe description of manual attached to the above rat cDNA library, whichwas transformed into Escherichia coli to make an Escherichia colilibrary. Then, this Escherichia coli library was used to make a membranefor hybridization according to a method described in Recombinant DNAExperiment (editted by J. R. Dillon, A Nasim, E. R. Nestmann, TokyoKagakudozin, 1987), pp98 to 100. Further, this membrane was used toperform hybridization using DIG-High Prime DNA Labeling and DetectionStarter Kit I (manufactured by Boehringer Mannheim) and reagentsattached thereto and according to the protocol attached thereto. As aprobe, a DNA fragment having the nucleotide sequence shown by nucleotideNos. 639 to 1333 in SEQ ID; No. 2. As a result, two clones whichstrongly hybridize with the probes were obtained.

The nucleotide sequence of the DNA fragment harbored by the resultantclone was determined using Dye terminator cycle sequencing kit(manufactured by PE Applied Systems) and a DNA sequencer (DNA sequencer373S™ (manufactured by PE Applied Systems)). As a result, the nucleotidesequence shown by nucleotide Nos. 270 to 1636 in SEQ ID: No. 2 wasrevealed and it was found that the resultant two clones harbor the DNAfragments having the same nucleotide sequence. In addition, since atranslation initiation codon is missing in the determined base sequence,a PPO gene encoded in the cloned DNA fragment was found to lack a 5′upstream region containing a translation initiation codon.

Example 2 Cloning of a Full Length PPO Gene

In order to clone a 5′ upstream region of a PPO gene which is missing inthe PPO DNA fragment obtained in Example 1, a polymerase chain reactionwas performed using as a template a DNA extracted from a rat cDNAlibrary to amplify the DNA fragment. A reaction solution for polymerasechain reaction was prepared by taking 10 pmol of an oligobase having thenucleotide sequence shown by SEQ ID:No. 4, 10 pmol T3 primer(manufactured by TAKARA SHUZO CO., LTD), 0.5 μl TaKaRa LATaq(manufactured by TAKARA SHUZO CO., LTD), 5 μl 10XLA PCR buffer(manufactured by TAKARA SHUZO CO., LTD), eaeh 20 nmol of four kinds ofnucleotides (dATP, dCTP, dGTP, dTTP: manufactured by Clontech), and 10ng of phage cloned DNA in a 0.5 ml volume of a polymerase chain reactiontube and adding sterile distilled water to total 50 μl. Each step ofpolymerase chain reaction was carried out under the followingconditions: The first cycle comprising a denaturing step holding atemperature at 95° C. for 1 minute, an annealing step holding atemperature at 55° C. for 2 minutes, and an extension step with a DNApolymerase holding a temperature at 72° C. for 3 minutes was performedonce, and the second cycle comprising a denaturing step holding atemperature at 95° C. for 1 minute, an annealing step holding atemperature at 55° C. for 1.5 minutes, and an extension step with apolymerase holding a temperature at 72° C. for 2 minutes was performed34 times. After completion of the polymerase chain reaction, thereaction solution was filtered by a spin column (MicroSpin S-400HR™(manufactured by Pharmacia Biotech)) to purify the DNA fragmentsamplified by the polymerase chain reaction.

Next, 2 μt of the purified solution of the DNA fragment amplified by theabove polymerase chain reaction, 50 ng of linearized pCR2.1(manufactured by Invitrogen), 1 μl of Ligation buffer (manufactured byInvitrogen), and 4 Weiss units of T4 DNA Ligase (manufactured byInvitrogen) were taken into 1.5 ml microtube, a sterile distilled waterwas added thereto to total 10 μl to mix, a DNA ligation reaction wasperformed by holding a temperature at 14° C. for 16 hours to ligate theDNA fragment amplified by the polymerase chain reaction to pCR2.1. Aftercompletion of ligation reaction, a competent cell of Escherichia colistrain INV a F(manufactured by Invitrogen) was transformed with 1 μl ofthe reaction solution to select a strain which becameampicillin-resistant. Further, plasmid DNA was extracted from theselected strain and they were cut with restriction enzymes and analyzedon an agarose electrophoresis to select a plasmid in which about 1.4 kbpDNA fragment amplified by polymerase chain reaction was cloned.

The nucleotide sequence of the DNA fragment harbored by the selectedplasmid was determined using Dye terminator cycle sequencing kit (PEApplied Bio Systems) and a DNA sequencer 373S (manufactured by PEApplied Bio Systems). The determined nucleotide sequence was analyzedand found that the cloned 1.4 kbp DNA fragment overlap with the DNAfragment obtained in Example 1 by about 1.1 kbp and contain about 150 bpupstream from translation initiation point.

The above plasmid having about 1.4 kbp DNA fragment was cut with arestriction enzyme, EcoRI and HincII (both manufactured by TAKARA SHUZOCO., LTD) to recover about 550 bp fragment. On the other hand, theplasmid harbored by the clone obtained in Example 1 was cut with arestriction enzyme, EcoRI and HincII (both manufactured by TAKARA SHUZOCO., LTD) and its 5′ terminal was dephosphorylated with calf intestinealkaline phosphatase (manufactured by TAKARA SHUZO CO., LTD).

The aforementioned two DNA fragments were taken in a microtube, andligated using a DNA ligation kit (manufactured by TAKARA SHUZO CO.,LTD). Escherichia coli HB101 competent cell (manufactured by TAKARASHUZO CO., LTD) was transformed with the resultant reaction solution andselected a strain which became ampicillin-resistant. The plasmidharbored by the selected strain was cut with restriction enzymes EcoRIand XhoI (both manufactured by TAKARA SHUZO CO., LTD), and analyzed onan agarose gel electrophoresis to select a plasmid into which about 1.7kbp DNA fragment is cloned.

The nucleotide sequence of the DNA fragment harbored by the resultantclone was determined using Dye terminator cycle sequencing kit(manufactured by PE Applied Bio Systems) and a DNA sequencer 373S(manufactured by PE Applied Bio Systems). As a result, the nucleotidesequence shown by SEQ ID: No.2 was revealed and the cloned DNA fragmentwas found to contain a full length structural gene (1431 bp) encoding arat-derived PPO.

Example 3 Analysis of PPO Amino Acid Sequence

The nucleotide sequence of the rat-derived PPO gene cDNA determined inExample 2 was analyzed to translate the amino acid sequence usinggenetic analyzing software (GENETYX: manufactured by SDC). As a result,a protein encoded by the cloned rat-derived PPO gene cDNA was found tobe composed of 477 amino acid residues and its amino acid sequence wasthat shown by SEQ ID: No. 1.

Example 4 Construction of a Vector for Expressing a Rat PPO Gene inEscherichia coli

An oligonucleotide having the nucleotide sequence shown by SEQ ID: No. 5and that having the nucleotide sequence shown by SEQ ID: No. 6 wereprepared. The oligonucleotides were synthesized using a DNA synthesizer(PE Applied Systems: Model 394 DNA/RNA Synthesizer) and using a solventfor Model 394 DNA/RNA Synthesizer as a solvent (PE Applied Systems) andusing as a DNA synthesizing reagent phosphoamidite reagentscorresponding to adenine, cyctosine, guanine and thymidine (PE AppliedSystems). The synthesized oligonucleotides were purified by anoligonucleotide purifying cartridge (PE Applied Systems: OPC cartridge)and dried under reduced pressure to prepare oligonucleotides.

A polymerase chain reaction was performed using as a template a fulllength gene cDNA encoding the rat-derived PPO obtained in Example 2 andas a primer an oligonucleotide having the nucleotide sequence shown bySEQ ID: No. 5 and that having the nucleotide sequence shown by SEQ ID:No. 6 to amplify the about 1.5 kbp DNA fragment encoding PPO. A reactionsolution in the polymerase chain reaction was prepared by taking 10 pmolof an oligonucleotide having the nucleotide sequence shown by SEQ ID;No. 5, 10 pmol of an oligonucleotide having the nucleotide sequenceshown by SEQ ID: No. 6, 0.5 μl of long-amplifying Taq DNA polymerase(TaKaRa LA Taq manufactured by TAKARA SHUZO CO., LTD), 5.0 μl 10× LA PCRbuffer (manufactured by TAKARA SHUZO CO., LTD), each 20 nmol of fourkinds of nucleotides (dATP, dCTP, dGTP and dTTP; manufactured byClontech), and 10 ng of a plasmid containing full length cDNA of the ratPPO obtained in Example 2 in a 0.5 ml volume of a polymerase chainreaction tube and adding sterile distilled water to total 50 μl. Eachstep in the polymerase chain reaction was performed under the followingconditions: The first cycle comprising a denaturing step holding atemperature at 95° C. for 1 minute, an annealing step holding atemperature at 55° C. for two minutes, and extension step with a DNApolymerase holding a temperature at 72° C. for three minutes wasperformed once, and the second cycle comprising a denaturing stepholding a temperature at 95° C. for 1 minute, an annealing step holdinga temperature at 55° C. for 1.5 minutes, and an extension step holding atemperature at 72° C. for 2 minutes was performed 34 times.

After the polymerase chain reaction, the DNA fragment amplified by thepolymerase chain reaction was purified by filtering the reactionsolution with a spin column (MicroSpin S-400HR manufactured by PharmaciaBiotech). A terminus of this DNA fragment was cut with restrictionenzymes SacII and SmaI. On the other hand, pBluescript II SK+(manufactured by Stratagene) was cut with restriction enzymes SacII andSmaI (both manufactured by TAKARA SHUZO CO., LTD.) and the 5′ end wasdephosphorylated with calf intestine alkaline phosphatase (manufacturedby TAKARA SHUZO CO., LTD).

The aforementioned two DNA fragments were taken in a microtube, andligated using a DNA ligation kit (manufactured by TAKARA SHUZO CO.,LTD). A competent cell of Escherichia coli JM109 (manufactured by TAKARASHUZO CO., LTD) was transformed with the resultant reaction solution toselect a strain which became ampicillin-resistant and a plasmid DNA wasprepared from the ampicillin-resistant strain to obtain a vector forexpressing rat PPO gene in Escherichia coli.

Example 5 Complementation Experiment of Escherichia coli hemg-deficientStrain With Rat PPO Gene

The expression vector obtained in Example 4 was introduced intoEscherichia coli PPO gene (hemG locus)-deficient mutant strain BT3,which was spread-inoculated on LB agar medium containing kanamycin atfinal concentration of 10 μg/ml and ampicillin at final concentration of50 μg/ml to culture at 37° C. for 2 days. BT3 strain became lessproliferative and formed only small colonies, while a strain having theintroduced vector for expressing rat PPO gene formed relatively largecolonies on the LB plate.

Example 6 Test on the Ability of a Compound to Inhibit the Rat PPOActivity

An overnight cultured bacterial solution of Escherichia coli strain BT3into which a introduced vector for expressing the rat PPO gene obtainedin Example 5 is diluted to turbidity(OD₆₀₀) of 1.0.0.05% medium volumeequivalent amount of this diluted bacterial solution is inoculated onYPT liquid medium containing no various test compounds and also isinoculated on YPT liquid medium containing various compounds at avariety of concentrations to culture by shaking at 37° C. and turbidity(OD₆₀₀) 16 hours after culturing is measured. The concentration at which50% growth inhibition by various test compounds is seen is calculated byadopting turbidity of the system having no addtion of test compound as100%. By comparing the concentrations in various test compounds, the PPOinhibiting abilities of test compounds are determined.

Example 7 Obtaining of Escherichia coli for Expressing Rat PPO Gene,into Which lac Repressor Gene is Introduced

pBR322 was cut with restriction enzymes HindIII and AvaI (bothmanufactured by TAKARA SHUZO CO., LTD) to recover the about 1.4 kbfragment. Separately, pREP4 was cut with restriction enzymes HindIII andAvaI (both manufactured by TAKARA SHUZO CO., LTD) to recover the about2.4 kb fragment and its 5′end was dephosphorylated with calf intestinealkaline phosphatase (manufactured by TAKARA SHUZO CO., LTD).

The aforementioned two DNA fragments were taken in a microtube andligated using a DNA ligation kit (manufactured by TAKARA SHUZO CO.,LTD). A competent cell (manufactured by TAKARA SHUZO CO., LTD) ofEscherichia coli strain JM109 was transformed with the resultantreaction solution to select a strain which became kanamycin andtetracyclin-resistant and a plasmid DNA was prepared from the kanamycinand tetracyclin-resistant strain to obtain a vector for expressing lacrepressor. The vector was introduced into Escherichia coli strain BT3 toselect a strain which became kanamycin, tetracyclin andampicillin-resistant. Further, the expression vector obtained in Example4 was introduced into the tetracyclin-resistant strain to select astrain which became kanamycin and tetracyclin- and ampicillin-resistantto obtain Escherichia coli strain BT3 into which the rat PPO gene andlac repressor gene were introduced.

Example 8 Assaying of the Ability of a Compound to Inhibit the PPOActivity Using Escherichia coli Strain BT3 for Expressing Rat PPO Gene,into Which lac Repressor is Introduced

An overnight cultured bacterial solution of Escherichia coli strain BT3for expressing a rat PPO gene, into which lac repressor was introduced,obtained in Example 7 was diluted to turbidity (OD₆₀₀) of 0.5. 0.1%medium volume equivalent amount of this diluted bacterial solution wasinoculated on YPT liquid medium containing no test compound and also wasinoculated on YPT liquid medium containing various test compounds atvarious concentrations to culture by shaking at 37° C. and turbidity(OD₆₀₀) 20 hours after culturing was measured. The concentration atwhich 50% growth inhibition by a compound having the structure shown inFIG. 4 was seen was calculated and found to be 0.11 ppm under thecondition that the growth rate of the transformant is 100% in the systemof no addition of test compound.

Example 9 Cloning of Chlamydomonas reinhardtii PPO DNA Fragment

Chlamydomonas reinhardtii strain CC407 was obtained from ChlamydomonasGenetics Center (address: DCMB Group, Department of Botany, Box 91000,Duke University, Durham, N.C. 27708-1000, USA), and cultured in TAPliquid medium comprising 7 mM NH₄Cl, 0.4 mM MgSO₄.7H₂O, 0.34 mMCaCl₂.2H₂O, 25 mM potassium calcium, 0.5 mM Tris (pH 7.5), 1 ml/L Hutnertrace elements, and 1 ml/L glacial acetic acid (E. H. Harris, TheChlamydomonas Sourcebook, Academic Press, San Diego, 1989, pp576-577)under the light (200 μE/m²¹) for 5 days to obtain 200 ml of culturecontaining cells at early stationary prolifelation stage (1.0×10⁶cells/ml).

The whole RNA was prepared from the cells by performing the manipulationusing ISOGEN (Nippon gene) according to the attached manual. Further,poly(A) RNA was fractionated using BioMag mRNA Purification Kit(Perceptive Biosystems) by performing the manipulation according to theattached manual. From the resultant poly(A)RNA, a cDNA was synthesizedusing Marathon cDNA Amplification Kit (Clontech) by performing themanipulation according to the attached manual and this was used as atemplate for polymerase chain reaction.

An oligonucleotide having the nucleotide sequence shown by SEQ ID: No.11 and that having the nucleotide sequence shown by SEQ ID: 12 wereprepared as a primer for polymerase chain reaction. The oligonucleotideswere synthesized using a DNA synthesizer (PE Applied Systems: Model 394DNA/RNA Synthesizer) and using as a solvent for synthesis a solvent forModel 394 DNA/RNA Synthesizer (PE Applied Systems) and using as areagent for synthesizing DNA phosphoamidite reagents corresponding toadenine, cytosine, guanine and thymine (PE Applied Systems). Synthesizedoligonucleotides were purified with oligonucleotide purifying cartridge(PE Applied Systems: OPC cartridge) and dried under reduced pressure toprepare an oligonucleotide.

A polymerase chain reaction was performed by preparing a reactionsolution using Advantage cDNA PCR kit (Clontech) according to theattached manual and repeating once a cycle comprising 94° C. for 1minute and 70° C. for 4 minutes, four times a cycle comprising 94° C.for 10 seconds, then 70° C. for 4 minutes, five times a cycle comprising94° C. for 10 seconds, then 68° C. for 4 minutes, and 25 times a cyclecomprising 94° C. for 10 seconds, then 65° C. for 5 minutes, and analiquot of the reaction solution was subjected to agarose gelelectrophoresis to confirm that about 2 kbp amplified fragment isobtained. Further, the excess primers in the reaction solution wereremoved by performing the manipulation using a spin column (MicroSpinS400HR Pharmacia Biotech) according to the attached manual and themanipulation was performed using TA Cloning Kit (Invitrogen) accordingto the attached manual to clone the amplified fragment into pCR2.1plasmid.

The nucleotide sequence of the DNA fragment harbored by the resultantrecombined plasmid was determined using Dye terminator cycle sequencingkit (manufactured by PE Applied Bio Systems) and a DNA sequencer 373S(manufactured by PE Applied Bio Systems). As a result, the nucleotidesequence shown by SEQ ID; No. 10 was revealed and it was found to befull length cDNA encoding the amino acid sequence shown by SEQ ID: No. 9by the analysis using Genetic Analyzing software GENETYX (SDC).

Example 10 Construction of a Vector for Expressing a Chlamydomonasreinhardtii PPO Gene in Escherichia coli

An oligonucleotide having the nucleotide sequence shown by SEQ ID: No.13, in which a restriction enzyme SacI recognition sequence isintroduced at an end, and an oligonucleotide having the nucleotidesequence shown by SEQ ID: No. 14, in which a restriction enzyme SalIrecognition sequence is introduced at an end were prepared. Theoligonucleotides were synthesized using a DNA synthesizer (PE AppliedSystems: Model 394 DNA/RNA Synthesizer) and as a solvent for synthesis asolvent for Model 394 DNA/RNA Synthesizer (PE Applied Systems) and usingas a reagent for DNA synthesis a phosphoamidite reagent corresponding toadenine, cytosine, guanine, and thymine (PE Applied Systems). Thesynthesized oligonucleotides were purified by an oligonucleotidepurifying cartridge (PE Applied Systems: OPC cartridge) and dried underreduced pressure to prepare an oligonucleotide.

A polymerase chain reaction was performed using as a template a fulllength gene cDNA encoding PPO derived from Chlamydomonas reinhardtiiobtained in Example 9 and as a primer an oligonucleotide having thenucleotide sequence shown by SEQ ID: No. 13 and that having thenucleotide sequence shown by SEQ ID: No 14 to amplify the about 2 kbpDNA fragment encoding PPO. A reaction solution in the polymerase chainreaction was prepared by taking 10 pmol of an oligonucleotide having thenucleotide sequence shown by SEQ ID: No. 13, 10 pmol of anoligonucleotide having the nucleotide sequence shown by SEQ ID: No. 14,0.5 μl of long-amplifying Taq DNA polymerase (TaKaRa LA Taq manufacturedby TAKARA SHUZO CO., LTD), 5.0 μl of 10× LA PCR buffer (manufactured byTAKARA SHUZO CO., LTD), each 20 nmol of four kinds of nucleotides (dATP,dCTP, dGTP, dTTP: manufactured by Clontech), 10 ng of a plasmidcontaining full length cDNA of Chlamydomonas reinhardtii PPO obtained inExample 9 in 0.5 ml volume of a polymerase chain reaction tube andadding sterile distilled water to total 50 μl. Each step in thepolymerase chain reaction was performed under the following conditions:After the first cycle comprising a denaturing step holding a temperatureat 95° C. for 1 minute, an annealing step holding a temperature at 55°C. for 2 minutes, and an extension step with a DNA polymerase holding atemperature 72° C. for 3 minutes was performed once, the second cyclecomprising a denaturing step holding a temperature at 95° C. for 1minute, an annealing step holding a temperature at 55° C. for 15minutes, and an extension step holding a temperature at 72° C. for 2minutes was performed 34 times.

After the polymerase chain reaction, the DNA fragment amplified by thepolymerase chain reaction was purified by filtering the reactionsolution with MicroSpin S-400HR (manufactured by Pharmacia Biotech). Theend of this DNA fragment was cut with restriction enzymes SacI and SalI.On the other hand, the plasmid pUC 118 (manufactured by TAKARA SHUZOCO., LTD) was cut with restriction enzymes SacI and SalI (bothmanufactured by TAKARA SHUZO CO., LTD) and Send was dephosphorylatedwith calf intestine alkaline phosphatase (manufactured by TAKARA SHUZOCO., LTD).

The aforementioned two DNA fragments were taken in a microtube andligated using a DNA ligation kit (manufactured by TAKARA SHUZO CO.,LTD). A competent cell (manufactured by TAKARA SHUZO CO., LTD) ofEscherichia coli JM109 was transformed with the resultant reactionsolution to select a strain which became ampicillin-resistant and aplasmid DNA was prepared from the ampicillin-resistant strain to obtaina vector for expressing Chlamydomonas reinhardtii PPO gene.

Example 11 Complementation Experiment of Escherichia coli hemG-deficientStrain with Chlamydomonas reinhardtii PPO Gene

The expression vector obtained in Example 10 was introduced into PPOgene (hemG locus)-deficient mutant Escherichia coli strain BT3 describedin F. Yamamoto, H. Inokuchi, H. Ozeki, (1988) Japanese Journal ofGenetics, vol.63, pp237 to 249, which was spread-inoculated on LB agarmedium containing kanamycin to the final concentration of 10 μg/ml andampicillin to the final concentration of 50 μg/ml to culture at 37° C.for 2 days. Escherichia coli BT3 strain became less proliferative andformed small colonies, while a strain having introduced vector forexpressing Chlamydomonas reinhardtii PPO gene formed relatively largecolonies on the LB plate.

Example 12 Construction of a PPO Gene Expressing Vector for DirectIntroduction

In order to express a rat-derived PPO gene in a plant cell, a PPO geneexpressing vector for directly introducing a plant is constructed.

An oligonucleotide having the nucleotide sequence shown by SEQ ID: No. 7and that having the nucleotide sequence shown by SEQ ID: No.8 areprepared. The oligonucleotides are synthesized using a DNA synthesizer(PE Applied Systems: Model 394 DNA/RNA Synthesizer) and using as asolvent for DNA synthesis a solvent for Model 394 DMA/RNA Synthesizer(PE Applied Systems) and as a reagent for synthesis phosphoramiditereagents corresponding to adenine, cytosine, guanine and thymine (PEApplied Systems). The synthesized oligonucleotides are purified by anoligonucleotide purifying cartridge (PE Applied Systems: OPC cartridge)and dried under reduced pressure to prepare an oligonucleotide.

A polymerase chain reaction is performed using as a template a fulllength cDNA of the rat-derived PPO obtained in Example 2, and using as aprimer an oligonucleotide having the nucleotide sequence shown in SEQID: No. 7 and that having the nucleotide sequence shown in SEQ ID: No. 8to amplify the about 1.5 kbp DNA fragment encoding the rat PPO. Thepolymerase chain reaction is performed by adding 10 pmol of anoligonucleotide having the nucleotide sequence shown in SEQ ID: No. 7,10 pmol of an oligonucleotide having the nucleotide sequence shown inSEQ ID: No. 8, 0.5 μl of Advantage KlenTaq Polymerase Mix (manufacturedby Clontech), 2.5 μl of 10× KlenTaq PCR reaction buffer (manufactured byClontech), each 5 nmol of four kinds of nucleotides (dATP, dCTP, dGTP,dTTP: manufactured by Clontech), and 10 ng of the full length of cDNA ofthe rat-derived PPO obtained in Example 2 in a 0.2 ml volume of apolymerase chain reaction tube to total amount of 25 μl. Each step inthe polymerase chain reaction is performed under the followingconditions: After the first cycle comprising a denaturing step holding atemperature at 94° C. for 1 minute, and an annealing step and extensionstep with a DNA polymerase holding a temperature at 65° C. for 4 minutesis performed once, the second cycle comprising a denaturing step holdinga temperature at 94° C. for 30 seconds, and an annealing step andextension step with a DNA polymerase holding a temperature at 65° C. for4 minutes is performed 15 times. After completion of the polymerasechain reaction, the DNA fragment amplified in the polymerase chainreation is purified by filtering the reaction solution with a spincolumn (MicroSpin S-400HR manufactured by Pharmacia Biotech). After theend of this DNA fragment is made blunt with a DNA blunting kit(manufactured by TAKARA SHUZO CO., LTD), a phosphate group is added tothe 5′ end with T4 polynucleotide kinase (manufactured by TAKARA SHUZOCO., LTD).

On the other hand, after the GUS expression vector pBI221 derived frompUC19 (manufactured by Clontech) is cut with restriction enzymes SmaIand SacI (both manufactured by TAKARA SHUZO CO., LTD) to recover the 2.8kbp DNA fragment from which the GUS structural gene has been removed andits end is made blunt using a DNA blunting kit (manufactured by TAKARASHUZO CO., LTD), the DNA fragment is dephosphorylated with bacterialalkaline phosphatase (manufactured by TAKARA SHUZO CO., LTD).

The aforementioned two DNA fragments are taken in a microtube andligated using a DNA ligation kit (manufactured by TAKARA SHUZO CO.,LTD). A competent cell of Escherichia coli strain HB 101 (manufacturedby TAKARA SHUZO CO., LTD) is transformed with the resultant reactionsolution to select a strain which has become ampicillin-resistant.Further, a plasmid in which a coding region for the rat-derived PPO isinserted in a forward direction relative to the cauliflower mosaicvirus-derived 35S promoter and the nopaline synthase-derived terminatoris selected from plasmids contained in the selected strain, to obtain arat PPO gene expression vector for directly introducing into a plant.

Example 13 Construction of a PPO Gene Expression Vector for IndirectIntroduction

In order to express a rat-derived PPO gene to in a plant cell, a PPOgene expression vector for indirect introduction into a plant isconstructed.

An oligonucleotide having the nucleotide sequence shown by SEQ ID: No.7and an oligonucleotide having the nucleotide sequence shown by SEQ ID:No.8 are prepared. The oligonucleotides are synthesized using a DNAsynthesizer (PE Applied Systems: Model 394 DNA/RNA Synthesizer), using asolvent for synthesis a solvent for Model 394 DNA/RNA Synthesizer (PEApplied Systems) and using as a reagent for DNA synthesis phosphoamiditereagents corresponding to adenine, cytosine, guanine and thymine (PEApplied Systems). After the synthesized oligonucleotides are purifiedwith an oligonucleotide purifying cartridge (PE Applied Systems: OPCcartridge), they are dried under reduced pressure to prepare theoligonucleotides.

A polymerase chain reaction is performed using as a template the fulllength cDNA for the rat-derived PPO obtained in Example 2, and using anoligonucleotide having the nucleotide sequence shown in SEQ ID: No. 7and that having the base sequence shown in SEQ ID: No. 8 to amplify theabout 1.5 kbp DNA fragment encoding the rat-derived PPO. The polymerasechain reaction is performed by adding 10 pmol of an oligonucleotidehaving the nucleotide sequence shown by SEQ ID: No. 7, 10 pmol of anoligonucleotide having the nucleotide sequence shown by SEQ ID: No. 8,0.5 μl of Advantage KlenTaq Polymerase Mix (manufactured by Clontech),2.5 μl 10× KlenTaq PCR reaction buffer (manufactured by Clontech),each 5n mol of four kinds of nucleotides (dATP, dCTP, dGTP, dTTP, manufacturedby Clontech), and 10 ng of a DNA containing the full length cDNA for therat-derived PPO obtained in Example 2 to 0.2 μl volume of a polymerasechain reaction tube to total volume of 25 μl. Each step of thepolymerase chain reaction is performed as follows: After the first cyclecomprising a denaturing step holding a temperature at 94° C. for 1minute, and an annealing step and an extension step with a DNApolymerase holding a temperature at 65° C. for 4 minutes is performedonce, the second step comprising a denaturing step holding a temperatureat 94° C. for 30 seconds, an annealing step and an extension step with aDNA polymerase holding a temperature at 65 C for 4 minutes is performed15 times. After completion of the polymerase chain reaction, the DNAfragment amplified by polymerase chain reaction is purified by filteringthe reaction solution with MicroSpin S-400HR (manufactured by PharmaciaBiotech). After the end of this DNA fragment is made blunt with a DNAblunting kit (manufactured by TAKARA SHUZO CO., LTD), a phosphate groupis added to the 5′end with the T4 polynucleotide kinase (manufactured byTAKARA SHUZO CO., LTD).

On the other hand, after the pBIN 19-derived GUS expressing binaryvector pBI121 (manufactured by Clontech) is cut with restriction enzymesSmaI and SacI (both manufactured by TAKARA SHUZO CO., LTD) to recoverthe DNA fragment from which the GUS structural gene has been removed andits end is made blunt using a DNA blunting kit (manufactured by TAKARASHUZO CO., LTD), it is dephosphorylated with a bacterial alkalinephosphatase (manufactured by TAKARA SHUZO CO., LTD).

The aforementioned two DNA fragments are taken in a microtube andligated using a DNA ligation kit (manufactured by TAKARA SHUZO CO.,LTD). A competent cell of Escherichia coli strain HB 101 (manufacturedby TAKARA SHUZO CO., LTD) is transformed with the resultant reactionsolution to select a strain which has become kanamycin-resistant.Further, a plasmid in which a coding region for a rat PPO is inserted ina forward direction relative to the cauliflower mosaic virus-derived 35Spromoter and the nopaline synthase-derived terminator from plasmidscontained in the selected strain, to obtain the rat PPO gene expressionvector for indirect introduction into a plant.

Example 14 Construction of a Vector for Expressing a Chlamydomonasreinhardtii PPO Gene

In order to express a Chlamydomonas reinhardtii-derived PPO in a plantcell, a PPO gene expression vector for direct introduction into a plantis constructed.

The pCR2.1 plasmid harboring the amplified fragment of the cDNA for theChlamydoinonas reinhardtii-derived PPO gene obtained in Example 9 isdigested with restriction enzymes NotI and SpeI (both manufactured byTAKARA SHUZO CO., LTD) to obtain the about 2 kbp Chlamydomonasreinhardtii-derived PPO gene cDNA fragment having cohesive ends for NotIand SpeI at its end. On the other hand, pBluescriptII KS+ (manufacturedby Stratagene) is cut with restriction enzymes NotI and SpeI (bothmanufactured by TAKARA SHUZO CO., LTD) and the 5′end is dephosphorylatedwith calf intestine alkaline phosphatase (manufactured by TAKARA SHUZOCO., LTD). The two DNA fragments are taken in a microtube and ligatedusing a DNA ligation kit (manufactured by TAKARA SHUZO CO., LTD). Acompetent cell of Escherichia coli strain HB 101 (manufactured by TAKARASHUZO CO., LTD) is transformed with the resultant reaction solution toselect a strain which has become ampicillin-resistant. Further, aplasmid contained in the selected strain is selected to obtain a clonein which the Chlamydomonas reinhardtii-derived PPO gene cDNA is insertedinto the plasmid pBluescriptII KS+.

The resultant plasmid is digested with restriction enzymes BamHI andSacI (both manufactured by TAKARA SHUZO CO., LTD) to obtain the about 2kbp Chlamydomonas reinhardtii-derived PPO gene cDNA fragment havingcohesive ends for BamHI and SacI at its ends. On the other hand, pBI221and pBI121 (both manufactured by Clontech) are cut with restrictionenzymes BamHI and SacI (both manufactured by TAKARA SHUZO CO., LTD),respectively, to obtain a vector fragment from which a β-glucuronidasegene has been removed and the 5′end is dephosphorylated with the calfintestine alkaline phosphatase (manufactured by TAKARA SHUZO CO., LTD).The Chlamydomonas reinhardtii-derived PPO gene cDNA fragment and the twoDNA fragments of the vector fragment of pBI221 or pBI121 are taken in amicrotube and ligated using a DNA ligation kit (manufactured by TAKARASHUZO CO., LTD). A competent cell of Escherichia coli strain HB 101(manufactured by TAKARA SHUZO CO., LTD) is transformed with theresultant reaction solution and a strain which has becomeampicillin-resistant, in the case of ligation with the pBI221 vectorfragment and, a plasmid which has become kanamycin-resistant, in thecase of ligation with pBI121 vector fragment are selected. Further, aplasmid contained in the selected strain is select to obtain a plasmidfor direct introduction in which the Chlamydomonas reinhardtii-derivedPPO gene cDNA fragment is inserted into the pBI221 vector fragment or aplasmid for indirect introduction in which the Chlamydomonasreinhardtii-derived PPO gene cDNA fragment is inserted into the pBI121vector fragment.

Example 15 Production of a Transformed Plant in Which a Rat orChlamydomonas reinhardtii-derived PPO Expression Vector is Introduced

The rat PPO gene expression vector for indirect introduction or theChlamydomonas reinhardtii PPO gene expression vector for indirectinduction obtained in Example 13 or Example 14 is transferred intoAgrobacterium tumefaciens LBA4404 by a binary vector method(manufactured by Clontech: GUS Gene Fusion Ststem). A sterile-culturedtobacco leaf is infected with this bacterial strain according to PlaneGene Manipulation Manual (author: Uchimiya, Kodansha Scientific, 1990)to obtain a transformed tobacco. Similarly, a sterile-cultured carrotseedling petiole is infected therewith according to a method describedin N. Pawlicki et al., (1992) Plant Cell, Tissue and Organ Culture,vol.31, pp.129 to 139 to obtain a transformed carrot.

Similarly, a sterile-cultured pea seedling epicotyl or cotyledon isinfected therewith according to a method described in J.Puonti-Kaerlaset al., (1990) Theoretical and Applied Genetics, vol.80, pp.246 to 252to obtain a transformed pea.

Further, the rat PPO gene expression vector for direct introduction orChlamydomonas reinhardtii PPO gene expression vector for directintroduction obtained in Example 12 or Example 15 is introduced intosoybean adventitious embryo with a particle gun according to a methoddescribed in JP-A-3-291501 (laid-opened) to obtain a transformedsoybean. Similarly, it is introduced into a sterile-cultured riceimmature scutellum with a particle gun according to a method describedin Ikushugakkaishi, vol.44, suppl.No.1, pp.66, (1994)(author: Shimada,et al.) to obtain a transformed rice. Similarly, it is introduced into asterile-cultured wheat immature scutellum with a particle gun accordingto the conventional method described in Ikushugakkaishi, vol.44, suppl.No.1, pp.57 (1995) (author: Takumi, et al.) to obtain a transformedwheat. Similarly, it is introduced into a sterile-cultured barleyimmature scutellum with a particle gun according to a method describedin Ikushugakkaishi, vol.44, suppl. No. 1, pp.67 (1995)(author: Hagio, etal.) to obtain a transformed barley. Similarly, it is introduced into acorn adventitious embryo with a particle gun according to a methoddescribed in M. E. Fromm et al., (1990) BIO/TECHNOLOGY, vol.8, pp.833 to839 to obtain a transformed corn.

The present invention makes it possible to provide a method forevaluating the ability of a compound to inhibit the PPO activity withsimplicity.

Brief Explanation of the Sequence

1. SEQ ID: No.1

Shows the amino acid sequence of a mitochondrial-type PPO encoded in acDNA for a rat-derived PPO gene.

2. SEQ ID: No.2

Shows the nucleotide sequence of a cDNA clone for a rat-derived PPOgene.

3. SEQ ID: No.3

Shows the nucleotide sequence of an oligonucleotide used for amplifyinga DNA fragment containing a partial nucleotide sequence of a rat-derivedPPO gene.

4. SEQ ID: No.4

Shows the nucleotide sequence of an oligonucleotide used for amplifyinga DNA fragment containing a partial nucleotide sequence of a rat-derivedPPO gene.

5. SEQ ID: No. 5

Shows the nucleotide sequence of a primer used for constructing a vectorexpressing a rat-derived PPO gene in Escherichia coli.

6.SEQ ID:No.6

Shows the nucleotide sequence of a primer used for constructing a vectorexpressing a rat-derived PPO gene in Escherichia coli.

7. SEQ ID: No. 7

Shows the nucleotide sequence of a primer used for constructing arat-derived PPO gene expression vector for direct introduction and arat-derived PPO expression vector for indirect introduction.

8. SEQ ID: No.8

Shows the nucleotide sequence of a primer used for constructing arat-derived PPO gene expression vector for direct introduction and arat-derived PPO expression vector for indirect introduction.

9.SEQ ID: No.9

Shows the amino acid sequence of PPO encoded in a cDNA for Chlamydomonasreinhardtii-derived PPO gene.

10. SEQ ID: No.10

Shows the nucleotide sequence of a cDNA clone for Chlamydomonasreinhardtii-derived PPO gene.

11. SEQ ID: No.11

Shows the nucleotide sequence of an oligonucleotide used for amplifyinga DNA fragment containing a Chlamydomonas reinhardtii-derived PPO gene.

12. SEQ ID: No.12

Shows the nucleotide sequence of an oligonucleotide used for amplifyinga DNA fragment containing a Chlamydomonas reinhardtii-derived PPO gene.

13. SEQ ID: No. 13

Shows the nucleotide sequence of a primer used for constructing a vectorexpressing a Chlamydomonas reinhardtii-derived PPO gene in Escherichiacoli.

14. SEQ ID: No.14

Shows the nucleotide sequence of a primer used for constructing a vectorfor expressing a Chlamydomonas reinhardtii-derived PPO gene inEscherichia coli.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 14 <210> SEQ ID NO 1 <211>LENGTH: 477 <212> TYPE: PRT <213> ORGANISM: Rattus norvegicus <400>SEQUENCE: Met Ala Arg Thr Val Ile Val Leu Gly Gly Gly Ile Ser Gly LeuAla 1 5 10 15 Ala Ser Tyr His Leu Thr Arg Ser Pro Ser Pro Pro Lys ValIle Leu 20 25 30 Val Glu Gly Ser Lys Arg Leu Gly Gly Trp Ile Arg Ser ValArg Gly 35 40 45 Ser Asp Gly Ala Ile Phe Glu Leu Gly Pro Arg Gly Ile ArgPro Ala 50 55 60 Gly Ala Leu Gly Ala Arg Thr Leu Leu Leu Val Ser Glu LeuGly Leu 65 70 75 80 Glu Ser Glu Val Leu Pro Val Arg Gly Asp His Pro AlaAla Gln Asn 85 90 95 Arg Phe Leu Tyr Val Gly Gly Ala Leu His Pro Leu ProSer Gly Leu 100 105 110 Arg Gly Leu Leu Arg Pro Ser Pro Pro Phe Ser LysPro Leu Phe Trp 115 120 125 Ala Gly Leu Arg Glu Leu Thr Lys Pro Arg GlyLys Glu Pro Asp Glu 130 135 140 Thr Val His Ser Phe Ala Gln Arg Arg LeuGly Pro Glu Val Ala Ser 145 150 155 160 Leu Ala Met Asp Ser Leu Cys ArgGly Val Phe Ala Gly Asn Ser Gln 165 170 175 Glu Leu Ser Ile Arg Ser CysPhe Pro Ser Leu Phe Gln Ala Glu Gln 180 185 190 Thr His Gly Ser Met LeuLeu Gly Leu Leu Leu Gly Ala Gly Gln Thr 195 200 205 Pro Gln Pro Asn SerSer Leu Ile Arg Gln Ala Arg Ala Glu Arg Trp 210 215 220 Ser Gln Trp SerLeu Arg Gly Gly Leu Glu Met Leu Pro Gln Ala Leu 225 230 235 240 His AsnTyr Leu Thr Ser Lys Gly Val Thr Ile Leu Ser Gly Gln Pro 245 250 255 AlaCys Gly Leu Ser Leu Gln Pro Glu Gly His Trp Lys Val Ser Leu 260 265 270Gly Asp Ser Ser Leu Glu Ala Asp His Ile Ile Ser Thr Ile Pro Ala 275 280285 Ser Val Leu Ser Lys Leu Leu Pro Ala Glu Ala Ala Pro Leu Ala His 290295 300 Ile Leu Ser Thr Ile Gln Ala Val Ser Val Ala Val Val Asn Leu Gln305 310 315 320 Tyr Lys Gly Ala Cys Leu Pro Val Gln Gly Phe Gly His LeuVal Pro 325 330 335 Ser Ser Glu Asp Pro Thr Val Leu Gly Ile Val Tyr AspSer Val Ala 340 345 350 Phe Pro Glu Gln Asp Gly Asn Pro Pro Gly Leu ArgLeu Thr Val Met 355 360 365 Leu Gly Gly Tyr Trp Leu Gln Lys Leu Lys AlaAsn Gly His Glu Leu 370 375 380 Ser Pro Glu Leu Phe Gln Arg Ala Ala GlnGlu Ala Ala Ala Thr Gln 385 390 395 400 Leu Gly Leu Lys Glu Gln Pro SerHis Cys Leu Val His Leu His Lys 405 410 415 Asn Cys Ile Pro Gln Tyr ThrLeu Gly His Trp Gln Lys Leu Asp Ser 420 425 430 Ala Leu Gln Phe Leu ThrAla Gln Arg Leu Pro Leu Thr Leu Ala Gly 435 440 445 Ala Ser Tyr Glu GlyVal Ala Val Asn Asp Cys Ile Glu Ser Gly Arg 450 455 460 Gln Ala Ala IleAla Val Leu Gly Thr Glu Ser Asn Ser 465 470 475 <210> SEQ ID NO 2 <211>LENGTH: 1638 <212> TYPE: DNA <213> ORGANISM: Rattus norvegicus <220>FEATURE: <222> LOCATION: (143)...(1576) <400> SEQUENCE: 2 cgtacacgcgcgttttgcat tagttgctca ttaatcagta agtgcccaga ggtggggtac 60 gggacccgtggggtttctgc agttgtaaag cagggtgcct cccgttctcc tggggtatct 120 cgactttcccccaggcctta cg atg gcc cgg act gtg ata gtg ctt ggc gga 172 Met Ala ArgThr Val Ile Val Leu Gly Gly 1 5 10 ggt atc agc gga ttg gcc gca agt tatcat ctg acc cga agc ccc agt 220 Gly Ile Ser Gly Leu Ala Ala Ser Tyr HisLeu Thr Arg Ser Pro Ser 15 20 25 cct cct aag gtg atc tta gtg gag ggc agcaaa cgt ttg gga ggc tgg 268 Pro Pro Lys Val Ile Leu Val Glu Gly Ser LysArg Leu Gly Gly Trp 30 35 40 atc cgt tca gtc cga gga tca gat ggt gcg atcttt gaa ctt gga cct 316 Ile Arg Ser Val Arg Gly Ser Asp Gly Ala Ile PheGlu Leu Gly Pro 45 50 55 cga gga att agg ccg gct gga gcc ctg gga gcc cggacc ctg ctc ctg 364 Arg Gly Ile Arg Pro Ala Gly Ala Leu Gly Ala Arg ThrLeu Leu Leu 60 65 70 gtt tct gaa ctt ggc ttg gaa tcc gaa gtc ttg cct gtccga ggg gat 412 Val Ser Glu Leu Gly Leu Glu Ser Glu Val Leu Pro Val ArgGly Asp 75 80 85 90 cat cca gct gcc cag aac cgg ttc ctg tat gta ggc ggtgcc ctg cac 460 His Pro Ala Ala Gln Asn Arg Phe Leu Tyr Val Gly Gly AlaLeu His 95 100 105 ccc cta ccc tct ggc ctc agg ggg cta ctt cgt cct tcaccc ccc ttc 508 Pro Leu Pro Ser Gly Leu Arg Gly Leu Leu Arg Pro Ser ProPro Phe 110 115 120 tca aaa cct cta ttt tgg gct gga ctg agg gag ttg acgaag ccc agg 556 Ser Lys Pro Leu Phe Trp Ala Gly Leu Arg Glu Leu Thr LysPro Arg 125 130 135 ggc aaa gag cct gat gag act gtg cac agt ttt gcc cagcgc cgc ctt 604 Gly Lys Glu Pro Asp Glu Thr Val His Ser Phe Ala Gln ArgArg Leu 140 145 150 gga cct gag gtg gcg tct ctg gct atg gac agc ctt tgcaga gga gtg 652 Gly Pro Glu Val Ala Ser Leu Ala Met Asp Ser Leu Cys ArgGly Val 155 160 165 170 ttt gct ggc aac agc caa gag ctc agc atc cgg tcctgc ttt ccc agt 700 Phe Ala Gly Asn Ser Gln Glu Leu Ser Ile Arg Ser CysPhe Pro Ser 175 180 185 ctc ttc caa gct gaa caa acc cac ggg tcc atg ttactg ggg ctg ctg 748 Leu Phe Gln Ala Glu Gln Thr His Gly Ser Met Leu LeuGly Leu Leu 190 195 200 ctg ggg gca ggg caa act cca cag ccc aat tcc tcatta att cgt cag 796 Leu Gly Ala Gly Gln Thr Pro Gln Pro Asn Ser Ser LeuIle Arg Gln 205 210 215 gcc cgc gct gag cga tgg agt cag tgg tca ctc cgtgga ggg ctg gag 844 Ala Arg Ala Glu Arg Trp Ser Gln Trp Ser Leu Arg GlyGly Leu Glu 220 225 230 atg ttg ccc cag gcc ctt cat aac tac cta aca agtaaa ggg gtc act 892 Met Leu Pro Gln Ala Leu His Asn Tyr Leu Thr Ser LysGly Val Thr 235 240 245 250 atc ctc agt ggt cag cca gcc tgc ggg ctc agcctt cag cca gaa ggg 940 Ile Leu Ser Gly Gln Pro Ala Cys Gly Leu Ser LeuGln Pro Glu Gly 255 260 265 cac tgg aag gtg tct cta ggg gac agc agt ctggag gct gac cac att 988 His Trp Lys Val Ser Leu Gly Asp Ser Ser Leu GluAla Asp His Ile 270 275 280 ata agc acc att cca gct tca gtg ctc agc aagctg ctc cct gcc gag 1036 Ile Ser Thr Ile Pro Ala Ser Val Leu Ser Lys LeuLeu Pro Ala Glu 285 290 295 gct gca cct ctg gct cac atc ctg agt acc atccaa gct gtg tct gtg 1084 Ala Ala Pro Leu Ala His Ile Leu Ser Thr Ile GlnAla Val Ser Val 300 305 310 gcc gtg gtg aat ctg cag tac aaa gga gct tgtctg cct gtg cag gga 1132 Ala Val Val Asn Leu Gln Tyr Lys Gly Ala Cys LeuPro Val Gln Gly 315 320 325 330 ttt gga cat ctg gtg cca tcc tca gaa gacccg acc gtc ctg gga atc 1180 Phe Gly His Leu Val Pro Ser Ser Glu Asp ProThr Val Leu Gly Ile 335 340 345 gtg tat gac tcg gtt gct ttt cct gag caggat ggg aac ccc cca ggc 1228 Val Tyr Asp Ser Val Ala Phe Pro Glu Gln AspGly Asn Pro Pro Gly 350 355 360 ctc aga ctg act gtg atg ttg gga ggt tactgg tta cag aag ctg aaa 1276 Leu Arg Leu Thr Val Met Leu Gly Gly Tyr TrpLeu Gln Lys Leu Lys 365 370 375 gcc aat ggc cat gaa ttg tct cca gag ctattc caa cga gca gca cag 1324 Ala Asn Gly His Glu Leu Ser Pro Glu Leu PheGln Arg Ala Ala Gln 380 385 390 gaa gcg gct gcc aca cag tta gga ctg aaagag caa cca agc cat tgc 1372 Glu Ala Ala Ala Thr Gln Leu Gly Leu Lys GluGln Pro Ser His Cys 395 400 405 410 ttg gtc cat cta cac aaa aac tgt atccct cag tat aca cta ggc cac 1420 Leu Val His Leu His Lys Asn Cys Ile ProGln Tyr Thr Leu Gly His 415 420 425 tgg caa aaa cta gac tca gct ctg caattc ctg acg gcc cag agg ttg 1468 Trp Gln Lys Leu Asp Ser Ala Leu Gln PheLeu Thr Ala Gln Arg Leu 430 435 440 ccc ctg act ttg gct ggg gcc tcc tatgag ggg gta gct gtc aat gac 1516 Pro Leu Thr Leu Ala Gly Ala Ser Tyr GluGly Val Ala Val Asn Asp 445 450 455 tgt ata gag agt ggg cgc cag gca gcaatt gct gtc ctg ggc aca gaa 1564 Cys Ile Glu Ser Gly Arg Gln Ala Ala IleAla Val Leu Gly Thr Glu 460 465 470 tcg aac agc tga cccccactctcctactcatg aaagtaaaag ttgatggagc 1616 Ser Asn Ser 475 ttgaaaaaaaaaaaaaaaaa aa 1638 <210> SEQ ID NO 3 <211> LENGTH: 28 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Designed oligonucleotide primer used for amplifying a DNAfragment containing a partial nucleotide sequence of a rat-derived PPOgene <400> SEQUENCE: 3 tttgcagagg agtgtttgct ggcaacag 28 <210> SEQ ID NO4 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide primerused for amplifying a DNA fragment containing a partial nucleotidesequence of a rat-derived PPO gene <400> SEQUENCE: 4 agccgcttcctgtgctgctc gttggaata 29 <210> SEQ ID NO 5 <211> LENGTH: 25 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Designed oligonucleotide primer used for constructing avector expressing a rat-derived PPO gene in E scherichia coli <400>SEQUENCE: 5 aggccttacc gcggcccgga ctgtg 25 <210> SEQ ID NO 6 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide primer usedfor constructing a vector expressing a rat-derived PPO gene in Escherichia coli <400> SEQUENCE: 6 taggagagcc cgggtcagat gttcg 25 <210>SEQ ID NO 7 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Designedoligonucleotide primer used for constructing a rat-derived PPO geneexpression vector for direct introduction and a rat-derived PPOexpression vector for indirect introd <400> SEQUENCE: 7 atggcccggactgtgatagt gcttg 25 <210> SEQ ID NO 8 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Designed oligonucleotide primer used for constructing arat-derived PPO gene expression vector for direct introduction and arat-derived PPO expression vector for indirect introd <400> SEQUENCE: 8ttcatgagta ggagagtggg ggtca 25 <210> SEQ ID NO 9 <211> LENGTH: 563 <212>TYPE: PRT <213> ORGANISM: Chlamydomonas reinhardtii CC-407 <400>SEQUENCE: 9 Met Met Leu Thr Gln Thr Pro Gly Thr Ala Thr Ala Ser Ser ArgArg 1 5 10 15 Ser Gln Ile Arg Ser Ala Ala His Val Ser Ala Lys Val AlaPro Arg 20 25 30 Pro Thr Pro Phe Ser Val Ala Ser Pro Ala Thr Ala Ala SerPro Ala 35 40 45 Thr Ala Ala Ala Arg Arg Thr Leu His Arg Thr Ala Ala AlaAla Thr 50 55 60 Gly Ala Pro Thr Ala Ser Gly Ala Gly Val Ala Lys Thr LeuAsp Asn 65 70 75 80 Val Tyr Asp Val Ile Val Val Gly Gly Gly Leu Ser GlyLeu Val Thr 85 90 95 Gly Gln Ala Leu Ala Ala Gln His Lys Ile Gln Asn PheLeu Val Thr 100 105 110 Glu Ala Arg Glu Arg Val Gly Gly Asn Ile Thr SerMet Ser Gly Asp 115 120 125 Gly Tyr Val Trp Glu Glu Gly Pro Asn Ser PheGln Pro Asn Asp Ser 130 135 140 Met Leu Gln Ile Ala Val Asp Ser Gly CysGlu Lys Asp Leu Val Phe 145 150 155 160 Gly Asp Pro Thr Ala Pro Arg PheVal Trp Trp Glu Gly Lys Leu Arg 165 170 175 Pro Val Pro Ser Gly Leu AspAla Phe Thr Phe Asp Leu Met Ser Ile 180 185 190 Pro Gly Lys Ile Arg AlaGly Leu Gly Ala Ile Gly Leu Ile Asn Gly 195 200 205 Ala Met Pro Ser PheGlu Glu Ser Val Glu Gln Phe Ile Arg Arg Asn 210 215 220 Leu Gly Asp GluVal Phe Phe Arg Leu Ile Glu Pro Phe Cys Ser Gly 225 230 235 240 Val TyrAla Gly Asp Pro Ser Lys Leu Ser Met Lys Ala Ala Phe Asn 245 250 255 ArgIle Trp Ile Leu Glu Lys Asn Gly Gly Ser Leu Val Gly Gly Ala 260 265 270Ile Lys Leu Phe Gln Glu Arg Gln Ser Asn Pro Ala Pro Pro Arg Asp 275 280285 Pro Arg Leu Pro Pro Lys Pro Lys Gly Gln Thr Val Gly Ser Phe Arg 290295 300 Lys Gly Leu Lys Met Leu Pro Asp Ala Ile Glu Arg Asn Ile Pro Asp305 310 315 320 Lys Ile Arg Val Asn Trp Lys Leu Val Ser Leu Gly Arg GluAla Asp 325 330 335 Gly Arg Tyr Gly Leu Val Tyr Asp Thr Pro Glu Gly ArgVal Lys Val 340 345 350 Phe Ala Arg Ala Val Ala Leu Thr Ala Pro Ser TyrVal Val Ala Asp 355 360 365 Leu Val Lys Glu Gln Ala Pro Ala Ala Ala GluAla Leu Gly Ser Phe 370 375 380 Asp Tyr Pro Pro Val Gly Ala Val Thr LeuSer Tyr Pro Leu Ser Ala 385 390 395 400 Val Arg Glu Glu Arg Lys Ala SerAsp Gly Ser Val Pro Gly Phe Gly 405 410 415 Gln Leu His Pro Arg Thr GlnGly Ile Thr Thr Leu Gly Thr Ile Tyr 420 425 430 Ser Ser Ser Leu Phe ProGly Arg Ala Pro Glu Gly His Met Leu Leu 435 440 445 Leu Asn Tyr Ile GlyGly Thr Thr Asn Arg Gly Ile Val Asn Gln Thr 450 455 460 Thr Glu Gln LeuVal Glu Gln Val Asp Lys Asp Leu Arg Asn Met Val 465 470 475 480 Ile LysPro Asp Ala Pro Lys Pro Arg Val Val Gly Val Arg Val Trp 485 490 495 ProArg Ala Ile Pro Gln Phe Asn Leu Gly His Leu Glu Gln Leu Asp 500 505 510Lys Ala Arg Lys Ala Leu Asp Ala Ala Gly Leu Gln Gly Val His Leu 515 520525 Gly Gly Asn Tyr Val Ser Gly Val Ala Leu Gly Lys Val Val Glu His 530535 540 Gly Tyr Glu Ser Ala Ala Asn Leu Ala Lys Ser Val Ser Lys Ala Ala545 550 555 560 Val Lys Ala 563 <210> SEQ ID NO 10 <211> LENGTH: 1838<212> TYPE: DNA <213> ORGANISM: Chlamydomonas reinhardtii CC-407 <220>FEATURE: <222> LOCATION: (2)...(1793) <400> SEQUENCE: 10 a atg atg ttgacc cag act cct ggg acc gcc acg gct tct agc cgg 46 Met Met Leu Thr GlnThr Pro Gly Thr Ala Thr Ala Ser Ser Arg 1 5 10 15 cgg tcg cag atc cgctcg gct gcg cac gtc tcc gcc aag gtc gcg cct 94 Arg Ser Gln Ile Arg SerAla Ala His Val Ser Ala Lys Val Ala Pro 20 25 30 cgg ccc acg cca ttc tcggtc gcg agc ccc gcg acc gct gcg agc ccc 142 Arg Pro Thr Pro Phe Ser ValAla Ser Pro Ala Thr Ala Ala Ser Pro 35 40 45 gcg acc gcg gcg gcc cgc cgcaca ctc cac cgc act gct gcg gcg gcc 190 Ala Thr Ala Ala Ala Arg Arg ThrLeu His Arg Thr Ala Ala Ala Ala 50 55 60 act ggt gct ccc acg gcg tcc ggagcc ggc gtc gcc aag acg ctc gac 238 Thr Gly Ala Pro Thr Ala Ser Gly AlaGly Val Ala Lys Thr Leu Asp 65 70 75 aat gtg tat gac gtg atc gtg gtc ggtgga ggt ctc tcg ggc ctg gtg 286 Asn Val Tyr Asp Val Ile Val Val Gly GlyGly Leu Ser Gly Leu Val 80 85 90 95 acc ggc cag gcc ctg gcg gct cag cacaaa att cag aac ttc ctt gtt 334 Thr Gly Gln Ala Leu Ala Ala Gln His LysIle Gln Asn Phe Leu Val 100 105 110 acg gag gct cgc gag cgc gtc ggc ggcaac att acg tcc atg tcg ggc 382 Thr Glu Ala Arg Glu Arg Val Gly Gly AsnIle Thr Ser Met Ser Gly 115 120 125 gat ggc tac gtg tgg gag gag ggc ccgaac agc ttc cag ccc aac gat 430 Asp Gly Tyr Val Trp Glu Glu Gly Pro AsnSer Phe Gln Pro Asn Asp 130 135 140 agc atg ctg cag att gcg gtg gac tctggc tgc gag aag gac ctt gtg 478 Ser Met Leu Gln Ile Ala Val Asp Ser GlyCys Glu Lys Asp Leu Val 145 150 155 ttc ggt gac ccc acg gct ccc cgc ttcgtg tgg tgg gag ggc aag ctg 526 Phe Gly Asp Pro Thr Ala Pro Arg Phe ValTrp Trp Glu Gly Lys Leu 160 165 170 175 cgc ccc gtg ccc tcg ggc ctg gacgcc ttc acc ttc gac ctc atg tcc 574 Arg Pro Val Pro Ser Gly Leu Asp AlaPhe Thr Phe Asp Leu Met Ser 180 185 190 atc ccc ggc aag atc cgc gcc gggctg ggc gcc atc ggc ctc atc aac 622 Ile Pro Gly Lys Ile Arg Ala Gly LeuGly Ala Ile Gly Leu Ile Asn 195 200 205 gga gcc atg ccc tcc ttc gag gagagt gtg gag cag ttc atc cgc cgc 670 Gly Ala Met Pro Ser Phe Glu Glu SerVal Glu Gln Phe Ile Arg Arg 210 215 220 aac ctg ggc gat gag gtg ttc ttccgc ctg atc gag ccc ttc tgc tcc 718 Asn Leu Gly Asp Glu Val Phe Phe ArgLeu Ile Glu Pro Phe Cys Ser 225 230 235 ggc gtg tac gcg ggc gac ccc tccaag ctg tcc atg aag gcg gcc ttc 766 Gly Val Tyr Ala Gly Asp Pro Ser LysLeu Ser Met Lys Ala Ala Phe 240 245 250 255 aac agg atc tgg att ctg gagaag aac ggc ggc agc ctg gtg gga ggt 814 Asn Arg Ile Trp Ile Leu Glu LysAsn Gly Gly Ser Leu Val Gly Gly 260 265 270 gcc atc aag ctg ttc cag gaacgc cag tcc aac ccg gcc ccg ccg cgg 862 Ala Ile Lys Leu Phe Gln Glu ArgGln Ser Asn Pro Ala Pro Pro Arg 275 280 285 gac ccg cgc ctg ccg ccc aagccc aag ggc cag acg gtg ggc tcg ttc 910 Asp Pro Arg Leu Pro Pro Lys ProLys Gly Gln Thr Val Gly Ser Phe 290 295 300 cgc aag ggc ctg aag atg ctgccg gac gcc att gag cgc aac atc ccc 958 Arg Lys Gly Leu Lys Met Leu ProAsp Ala Ile Glu Arg Asn Ile Pro 305 310 315 gac aag atc cgc gtg aac tggaag ctg gtg tct ctg ggc cgc gag gcg 1006 Asp Lys Ile Arg Val Asn Trp LysLeu Val Ser Leu Gly Arg Glu Ala 320 325 330 335 gac ggg cgg tac ggg ctggtg tac gac acg ccc gag ggc cgt gtc aag 1054 Asp Gly Arg Tyr Gly Leu ValTyr Asp Thr Pro Glu Gly Arg Val Lys 340 345 350 gtg ttt gcc cgc gcc gtggct ctg acc gcg ccc agc tac gtg gtg gcg 1102 Val Phe Ala Arg Ala Val AlaLeu Thr Ala Pro Ser Tyr Val Val Ala 355 360 365 gac ctg gtc aag gag caggcg ccc gcc gcc gcc gag gcc ctg ggc tcc 1150 Asp Leu Val Lys Glu Gln AlaPro Ala Ala Ala Glu Ala Leu Gly Ser 370 375 380 ttc gac tac ccg ccg gtgggc gcc gtg acg ctg tcg tac ccg ctg agc 1198 Phe Asp Tyr Pro Pro Val GlyAla Val Thr Leu Ser Tyr Pro Leu Ser 385 390 395 gcc gtg cgg gag gag cgcaag gcc tcg gac ggg tcc gtg ccg ggc ttc 1246 Ala Val Arg Glu Glu Arg LysAla Ser Asp Gly Ser Val Pro Gly Phe 400 405 410 415 ggt cag ctg cac ccgcgc acg cag ggc atc acc act ctg ggc acc atc 1294 Gly Gln Leu His Pro ArgThr Gln Gly Ile Thr Thr Leu Gly Thr Ile 420 425 430 tac agc tcc agc ctgttc ccc ggc cgc gcg ccc gag ggc cac atg ctg 1342 Tyr Ser Ser Ser Leu PhePro Gly Arg Ala Pro Glu Gly His Met Leu 435 440 445 ctg ctc aac tac atcggc ggc acc acc aac cgc ggc atc gtc aac cag 1390 Leu Leu Asn Tyr Ile GlyGly Thr Thr Asn Arg Gly Ile Val Asn Gln 450 455 460 acc acc gag cag ctggtg gag cag gtg gac aag gac ctg cgc aac atg 1438 Thr Thr Glu Gln Leu ValGlu Gln Val Asp Lys Asp Leu Arg Asn Met 465 470 475 gtc atc aag ccc gacgcg ccc aag ccc cgt gtg gtg ggc gtg cgc gtg 1486 Val Ile Lys Pro Asp AlaPro Lys Pro Arg Val Val Gly Val Arg Val 480 485 490 495 tgg ccg cgc gccatc ccg cag ttc aac ctg ggc cac ctg gag cag ctg 1534 Trp Pro Arg Ala IlePro Gln Phe Asn Leu Gly His Leu Glu Gln Leu 500 505 510 gac aag gcg cgcaag gcg ctg gac gcg gcg ggg ctg cag ggc gtg cac 1582 Asp Lys Ala Arg LysAla Leu Asp Ala Ala Gly Leu Gln Gly Val His 515 520 525 ctg ggg ggc aactac gtc agc ggt gtg gcc ctg ggc aag gtg gtg gag 1630 Leu Gly Gly Asn TyrVal Ser Gly Val Ala Leu Gly Lys Val Val Glu 530 535 540 cac ggc tac gagtcc gca gcc aac ctg gcc aag agc gtg tcc aag gcc 1678 His Gly Tyr Glu SerAla Ala Asn Leu Ala Lys Ser Val Ser Lys Ala 545 550 555 gca gtc aag gcctaa gcggctgcag cagtagcagc agcagcatcg ggctgtagct 1733 Ala Val Lys Ala 560ggtaaatgcc gcagtggcac cggcagcagc aattggcaag cacttggggc aagcggagtg 1793gaggcgaggg gggggctacc attggcgctt gctgggatgt gtagt 1838 <210> SEQ ID NO11 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide primerused for amplifying a DNA fragment containing a Chlamydomonasreinhardtii- derived PPO gene <400> SEQUENCE: 11 aatgatgttg acccagactcctgggacc 28 <210> SEQ ID NO 12 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Designed oligonucleotide primer used for constructing a vector forexpressing a Chlamydomonas reinhardtii- derived PPO gene in Escherichiacoli <400> SEQUENCE: 12 tactacacat cccagcaagc gccaatg 27 <210> SEQ ID NO13 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Designed oligonucleotide primerused for constructing a vector for expressing a Chlamydomonasreinhardtii- derived PPO gene in Escherichia coli <400> SEQUENCE: 13tcgagctcaa tgatgttgac ccagactcct gg 32 <210> SEQ ID NO 14 <211> LENGTH:32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Designed oligonucleotide primer used forconstructing a vector for expressing a Chlamydomonas reinhardtii-derived PPO gene in Escherichia coli <400> SEQUENCE: 14 ttgtcgactactacacatcc cagcaagcgc ca 32

What is claimed is:
 1. An isolated rat-derived nucleic acid encoding aprotein having protoporphyrinogen oxidase activity, wherein said nucleicacid comprises the nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding the amino acidsequence of SEQ ID NO:1; (b) a nucleotide sequence obtainable from a ratcDNA library which is amplified with a combination of PCR primers,wherein the forward primer is selected from the group consisting of SEQID NO: 5 and SEQ ID NO: 7, and the reverse primer is selected from thegroup consisting of SEQ ID NO:6 and SEQ ID NO:8, and wherein saidnucleotide sequence comprises the nucleotide sequence of apolynucleotide which is amplifiable with a combination of the PCRprimers of SEQ ID NO:3 and the PCR primer of SEQ ID NO:4; and (c) thenucleotide sequence of SEQ ID NO:
 2. 2. An isolated protoporphyrinogenoxidase nucleic acid comprising a nucleotide sequence encoding the aminoacid sequence of SEQ ID NO:
 1. 3. An isolated protoporphyrinogen oxidasenucleic acid comprising the nucleotide sequence of SEQ ID NO:
 2. 4. Anisolated DNA fragment which is a polynucleotide consisting of 700nucleotides or more of the polynucleotide of SEQ ID NO:2 or apolynucleotide encoding the polypeptide of SEQ ID NO:1.
 5. A vectorcomprising a protoporphyrinogen oxidase nucleic acid encoding a proteinhaving protoporphyrinogen oxidase activity, wherein said nucleic acidcomprises the nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence encoding the amino acid sequence of SEQ IDNO:1; (b) a nucleotide sequence obtainable from a rat cDNA library whichis amplified with a combination of PCR primers, wherein the forwardprimer is selected from the group consisting of SEQ ID NO: 5 and SEQ IDNO: 7, and the reverse primer is selected from the group consisting ofSEQ ID NO:6 and SEQ ID NO:8, and wherein said nucleotide sequencecomprises the nucleotide sequence of a polynucleotide which isamplifiable with a combination of the PCR primers of SEQ ID NO:3 and thePCR primer of SEQ ID NO:4; and (c) the nucleotide sequence of SEQ ID NO:2.
 6. A transformant produced by the introduction of the vector of claim5 into a host cell.
 7. The transformant according to claim 6, whereinthe host cell is a microorganism.
 8. The transformant according to claim6, wherein the host cell is a plant cell.